Activatable nanoprobes for intracellular drug delivery

ABSTRACT

An activatable nanoprobe is provided having a core component and an active agent associated with the core component via a bond configured to be cleaved upon exposure to an endogenous compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/493,815 filed Jun.11, 2012 which also claims priority to U.S. Provisional Application No.61,495,992 filed Jun. 11, 2011. The foregoing applications areincorporated herein in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

The work leading to this invention was partly supported by grants fromthe NIH Grant No. 2P01HL059412-11A1 and NSF Grant No. 0506560.Accordingly, the government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of activatable nanoprobes,and in certain embodiments, to multifunctional activatable nanoprobes.

BACKGROUND OF THE INVENTION

Cancer nanotechnology is a rapidly growing research area in nanomedicineinvolving disease diagnostics and therapy^(1,2). During the past decade,engineered nanoparticles integrated withmultimodality/multifunctionality have enabled imaging of cancer cellswith high sensitivity and demonstrated successful delivery of pre-loadedtherapeutic drugs in a targeted manner³⁻⁷. Multimodal nanoparticles thatare integrated with optical and magnetic imaging modalities^(8,9) havedemonstrated strong potential to facilitate pre-operative cancerdiagnosis by MRI and optical based imaging¹⁰⁻¹³, to provideintra-operative surgical guidance (by optically demarcating tumor tissuefrom healthy tissue), and to track tumor metastasis^(2,7,8).

Current nanoparticle technology allows for imaging of particles carryingtherapeutic drugs^(3,6,7,10,14,15). However, no activatable drugdelivery system has been reported to date that has demonstrated theability to directly confirm intracellular drug release upon reactionwith a cytosolic biomolecule. Up until now, challenges in designing andconstructing a nanoparticle integrating imaging, monitoring, andtherapeutic functionalities in a single unit have restricted thefabrication of such a nanoparticle system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows an optically activatable nanoprobe in an “off” state andan “on” state in accordance with an aspect of the present invention.

FIG. 1b is a TEM image of various activatable multifunctional/multimodalcomposite nanoprobes (MMCNPs) showing nearly spherical particles withirregular surface morphology indicative of the presence of satellitequantum dots (Qdots) on the IONPs (iron oxide nanoparticle core). Thesize of the MMCNPs ranges from 20 nm to 40 nm, due to polydispersity ofIONPs. The inset shows a high magnification TEM image of a single MMCNP.The IONP can be discerned in the image by its light grey contrast whilethe Qdots appeared with dark contrast on the IONP surface.

FIG. 2a shows a Qdot fluorescence emission spectra (excitationwavelength: 375 nm) measured as a function of time at 7.0 mM glutathione(GSH) concentration. These data show that full restoration of Qdotfluorescence occurs within one hour, after which no further increase inQdot fluorescence intensity is observed. The inset shows a plot of Qdotfluorescence intensity measured at the peak emission wavelength (582 nm)as a function of time. This plot illustrates that the fluorescenceintensity plateaus at 60 minutes. The (red) line of the plot is anon-linear fit to the data.

FIG. 2(b) shows a STAT-3 fluorescence emission spectra (excitationwavelength: 300 nm) measured as a function of time at 7.0 mM GSHconcentration. These data show that full restoration of STAT-3fluorescence occurs within one hour, after which no further increase inSTAT-3 fluorescence intensity is observed. The inset shows a plot ofSTAT-3 fluorescence intensity measured at the peak emission wavelength(430 nm) as a function of time. This plot illustrates that thefluorescence intensity plateaus at 60 minutes. The (red) line of theplot is a non-linear fit to the data.

FIG. 3a-3d includes phase-contrast (left panel) and correspondingepi-fluorescence (right panel) microscopy images of: (a) MDA-MB-231cells incubated with MMCNPs for 3 hours, showing significant Qdotfluorescence. This confirms extensive folate receptor mediated uptake ofthe MMCNPs; (b) MDA-MB-231 cells incubated with MMCNPs for 24 hours,again showing significant Qdot fluorescence that is similar to the 3hour incubation experiment. These data show that uptake of MMCNPs andsubsequent restoration of Qdot fluorescence (indicative of STAT-3release) occurs in less than 3 hours; (c) control experiment performedwith MDA-MB-231 cells to which no MMCNPs were added. As expected only aminor cellular autofluorescence is observed; (d) control experimentperformed with mouse thymus stromal epithelial cells, TE-71 incubatedwith MMCNPs for 24 hours. Similar to the control experiment shown inpanel (c) only background autofluorescence is observed at locations thatcorrespond to the locations of the cells, with no evidence of uptake ofMMCNPs by the TE-71 normal cells. This control experiment validates thatthe delivery of MMCNPs is highly targeted to MDA-MB-231 cells, whichover-express folate receptors.

FIGS. 4a-4c shows: (a) bright field; and (b) correspondingepi-luminescence laser microscopy images of MDA-MB-231 cells incubatedwith MMCNPs for 5 hours. The bright spots in the fluorescence imageindicate the location of aggregated Qdots, revealing that the MMCNPshave reacted with intra-cellular GSH. After this reaction, the GSHcoated Qdots are somewhat hydrophobic in nature leading to aggregationin the intra-cellular environment. Most of the aggregates as they appearin the bright field image (dark spots) and fluorescence image(corresponding bright spots) are localized near the cell membranebecause of their hydrophobic nature. (c) Normalized ensemblefluorescence emission spectra acquired by sample scanning laser confocalmicroscopy under 375 nm laser excitation. The ensembles are constructedby averaging fluorescence emission spectra obtained at differentlocations inside individual cells under illumination with a diffractionlimited laser spots (˜300 nm). Spectra were acquired at the location ofthe Qdot aggregates (red line) and the cellular regions without Qdots(autofluorescence, dark cyan line). As a control, the same experimentwas completed for Qdots in the “OFF state” (black line) and “ON state”(blue line) on glass substrates. Both the intra-cellular andextra-cellular “ON state” Qdots appear slightly red shifted with respectto the “OFF state” Qdots. In addition, the “ON state” Qdots aresignificantly broadened at the blue edge as well as the red edge of thespectra, possibly due to the presence of GSH on the Qdot surface. Thedifference in the appearance of the red shoulders in the intra-cellularand extra-cellular “ON state” Qdots is most likely due to the differencein the environment. The spectral feature around 500 nm in theintra-cellular “ON state” Qdot fluorescence emission ensemble spectrumis due the contribution of cellular autofluorescence.

FIGS. 5a-d show: (a) a schematic of agar phantom design. The agarphantom consists of four layers. From bottom to top these layers are:MDA-MB-231 cells embedded in agar (control), MMCNPs embedded in agar(control), MDA-MB-231 cells loaded with the MMCNPs embedded in agar(sample) and agar layer, respectively. (b) a digital photograph of agarphantom; and (c) a corresponding digital photograph of agar phantomunder 366 nm multiband UV irradiation. The MDA-MB-231 cells loaded withthe MMCNPs emit red fluorescence that is clearly visible to the nakedeye. Control cells do not show any detectable fluorescence emission. (d)an MRI image of agar phantom. The MDA-MB-231 cells loaded with theMMCNPs show strong MRI signal (indicated with false red color) incontrast with the control cells.

FIG. 6 shows results from a CyQuant® cell viability assay performed onhuman breast (MB-MDA-231) and pancreatic (Panc-1) cancer lines, andmouse thymus stromal epithelial line, TE-71. Cells were untreated(Control) or treated for 24 hours with MMCNPs to which no STAT-3inhibitor was attached (RII 61), STAT-3 inhibitor only (Compound), andMMCNPs (RII 64). Compared to untreated (Control), the viability of cellstreated with MMCNPs to which no STAT-3 is attached (RII 61) was attachedis not significantly different, indicating that the MMCNP itself do notcompromise cell viability. By contrast, the cells treated with 50 μMSTAT-3 inhibitor only (Compound) showed about a 15-20% decrease in cellviability, while cells treated with fully functional MMCNPs to whichSTAT-3 inhibitor was attached showed nearly 30% decrease in cellviability, even though the amount of STAT-3 inhibitor contained in theabout 5 μg of MMCNP administered in about 100 μL of cell media wasexpected to be less than the about 50 μM that was directly added to thecells in the other study. This key observation demonstrates theeffectiveness of the reported nanoparticle design in highly targeteddrug delivery to the cancer cells while maximizing cancer cell deathwith reduced amounts of drugs used compared to conventional approaches.Even though the MMCNP's consume much less STAT3 drug, the deliveryefficiency is dramatically increased, thus resulting in increasedtherapeutic efficiency while minimizing the potential for medical sideeffects due to the presence of excess free drug.

FIG. 7 is a high resolution TEM (HRTEM) image of a single MMCNP.Individual satellite Qdots on the IONP surface can be clearly identifiedby their single crystalline structure while the IONP (iron oxidenanoparticle core) in the HRTEM image is obscured by the satelliteQdots.

FIG. 8 shows FT-IR spectra of (a) dihydrolipoic acid, (b) lipoic acid,(c) dihydrolipoic acid coated IONPs, and (d) IONPs. The broad band at3200-3600 cm-1 indicates the surface hydroxyl group of the superparamagnetic IONPs (Figure S4d). The bands at about 3046 (O—H), about2934(—CH2-), about 1697 (C═O), about 1252 (O—H), and about 935 (OH) cm⁻¹were observed for dihydrolipoic acid and lipoic acid in (a) and (b)respectively. The presence of these characteristic bands into thespectra of dihydrolipoic acid coated IONPs (c) confirmed thedihydrolipoic acid coating on the surface of IONPs.

FIG. 9 shows normalized UV-Vis absorbance spectra of pure Qdots (red),IONP (black), Qdots attached to IONP (green) and MMCNPs (blue). AfterQdots are attached to IONPs, a large red shift of the absorptionspectrum is observed, indicating successful attachment of satelliteQdots to IONP core. The attachment of STAT-3 to form MMCNPs leads to anarrowing of the UV-Vis spectrum and the slight blue shift in comparisonto IONP-Qdot construct.

FIG. 10 shows a normalized emission spectra of pure Qdots (red), IONP(black), Qdots attached to IONP (green) and MMCNPs (blue). Only a blueshift of the MMCNPs with respect to the free Qdots and the IONP-Qdotconstruct is observed.

FIG. 11 shows a 3D plot of the Qdot fluorescence intensity recovery fromMMCNP (“OFF state”) as a function of time for GSH concentrations ofabout 2.8, about 4.2, about 5.7, and about 7.0 mM. Even at the lowestGSH concentration, the Qdot fluorescence recovers in approximately 1hour. Furthermore, the timescale of fluorescence recovery appears to beindependent of GSH concentration higher than about 1.4 mM. Since theintra-cellular concentration of GSH ranges from about 1 mM to about 15mM, it is expected that MMCNP uptaken by the cancer cells will releaseits cargo intracellularly (e.g., drug) within about an hour.

FIG. 12 is a TEM image of a plurality of IONPs showing polydispersity.Bar=20 nm.

FIG. 13 is a 3D reconstruction of MRI images recorded on agar phantom.This demonstrates the appearance of strong MRI signal from theMDA-MB-231 cells loaded with the MMCNPs (indicated with false redcolor).

FIG. 14 shows another embodiment of an optically activatable nanoprobe(DNCP) in accordance with an another aspect of the present invention.

FIG. 15 shows a plurality of STAT3 inhibitors for use with nanoparticlesin accordance with an aspect of the present invention.

FIG. 16 illustrates an exemplary NAC-modified STAT3 inhibitor.

FIG. 17 is a schematic illustrating sensing of cargo release by a Qdotcore due to intracellular GSH.

FIGS. 18a-b shows a nanoparticle having a CdS:Mn/ZnS quantum dot core,to which an S3I inhibitor drug will be covalently linked via a cleavabledisulfide bond linkage. (a) a lipid bilayer may be overcoated on thesurface of the S3I conjugated CdS:Mn/ZnS quantum dots. (b) the lipidbilayer may be further functionalized with folic acid to target cancercells that overexpress folate receptors, or with TAT peptide (acell-penetrating peptide).

FIGS. 19a-c show (a) an AFM image of zwitterionic (DC8,9PC) lipidvesicles and chemical structures of (b) DOTAP and (c) PtdEtn.

FIG. 20 shows the effect of an increase of EDTA concentration on thefluorescence intensity of GSH-Qdot.

FIG. 21 shows the effect of an increase of Cu ion concentration on thefluorescence intensity of GSH-Qdot.

FIG. 22 shows the effect of an increase of EDTA concentration on thefluorescence intensity of a NAC-Qdot.

FIG. 23 shows the effect of an increase of Cu ion concentration on thefluorescence intensity of a NAC-Qdot.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed new and unique activatablenanoprobes that may deliver active agents, and in some embodiments, mayalso be optically and magnetically imageable, targetable, and/or capableof reporting on intracellular drug release events. In one particularexemplary embodiment, an optically activatable nanoprobe is providedthat comprises an inorganic core, e.g., a super-paramagnetic iron oxidenanoparticle core (IONP), associated with satellite quantum dots(Qdots), e.g., CdS:Mn/ZnS quantum dots, where the Qdots themselves arefurther functionalized with an active agent, a targerting agent, and ahydrophilic dispersing agent. Advantageously, the Qdot luminescence isquenched in this nanoprobe (“OFF” state) due to combined electron/energytransfer mediated quenching processes involving IONP, targeting agentand active agents. Upon intracellular uptake, the nanoprobe is exposedto a cytosolic glutathione (GSH)-containing environment resulting inrestoration of the Qdot luminescence (“ON” state), which reports onuptake and drug release. Probe functionality was validated usingfluorescence and MR measurements, as well as in vitro studies usingcancer cells that overexpress folate receptors.

It is important to an understanding of the present invention to notethat all technical and scientific terms used herein, unless definedherein, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. The techniques employed herein arealso those that are known to one of ordinary skill in the art, unlessstated otherwise. Prior to setting forth the invention in detail and forpurposes of more clearly facilitating an understanding the invention asdisclosed and claimed herein, the following definitions are provided.

As used herein, the terms “about” and “approximately” as used hereinrefers to −values that are ±10% of the stated value.

As used herein, the term “active agent” includes any synthetic ornatural element or compound, which when introduced into a mammal causesa desired response, such as an optical or biological response.

As used herein, the term “activatable” refers to an agent capable ofbeing released from an associated substrate, e.g., core, upon exposureto a predetermined compound. For example and without limitation, anactivatable active agent may include an active agent that is releasedfrom a core component upon exposure to an endogenous molecule, such asglutathione, that cleaves a bond between the active agent and the corecomponent.

As used herein, the term “aptamer” refers to any oligonucleic acid orpeptide molecules that bind to a specific target molecule.

As used herein, the terms “chitosan” or “chitosan polymer” refer tochitosan (also known as poliglusam, deacetylchitin, poly-(D)glucosamine)and any derivatives thereof. The chitosan polymer is typically composedof a linear polysaccharide of randomly distributed β-(1-4)-linkedD-glucosamine (deacetylated unit) and/or N-acetyl-D-glucosamine(acetylated unit) units. The general terms “chitosan” or “chitosanpolymer” as used herein may also refer to chitosan or chitosan havingone or more molecules attached thereto, e.g., bonded, or conjugated,thereto, such as an imaging agent, a target-specific ligand, or abiologically active compound. Exemplary derivatives of chitosan includetrimethylchitosan (where the amino group has been trimethylated) orquaternized chitosan. Advantageously, chitosan has a plurality of aminefunctional groups, which as set forth below, may be utilized for theattachment of various agents thereto, such as imaging agents,target-specific ligands, and/or biologically active agents.

As used herein, “folate species” or “folate” refers to folate, folicacid, derivatives thereof, or analogs thereof.

As used herein, the terms “bonded,” “linked,” “labeled,” “attached,”“conjugated,” and variations thereof are intended to be usedinterchangeably and may refer to covalent, ionic, Van der Waals, orhydrogen bonding, for example.

As used herein, the term “hydrophilic” refers to any substance having anaffinity for water and tending to dissolve in, mix with, or swell in awater or aqueous medium.

As used herein, the term “hydrophobic” refers to any substance nothaving an affinity for water and tending not to dissolve in, mix with,or swell in a water or aqueous medium.

As used herein, the term “surfactant” refers to a wetting agent thatlowers the surface tension of a liquid, thereby allowing easierspreading and the lowering of the interfacial tension between twoliquids.

As used herein, the term “STAT” refers to signal transducers andactivators of transcription, which represent a family of proteins that,when activated by protein tyrosine kinases in the cytoplasm of the cell,migrate to the nucleus and activate gene transcription.

In accordance with one aspect of the present invention, there isprovided an activatable nanoprobe comprising a core component and anactive agent associated with the core component via a bond configured tobe cleaved upon exposure to an endogenous compound.

In accordance with one aspect, there is provided an activatablenanoprobe comprising a core component and an activatable active agentassociated with the core component. A lipid vesicle at least partiallyor fully encases the activatable agent and the core component.

In accordance with another aspect, there is an optically activatablenanoprobe comprising an inorganic core and a Qdot linked to theinorganic core. At least one ligand is linked to the Qdot. The one ormore ligands, which are typically electron-rich, are effective to reduceluminescence of the quantum dot when linked thereto. In this way, theQdot luminescence is quenched in this nanocomposite probe (“off state”)due to electron/energy transfer quenching processes between theinorganic core and the at least one ligand. In one embodiment, the atleast one ligand comprises at least one of a biologically active agent,a targeting agent, a hydrophilic dispersing agent, or combinationsthereof.

In accordance with another aspect, there is provided an opticallyactivatable nanoprobe for monitoring intracellular drug delivery. Thenanoprobe comprises a core component; and at least one ligand linked tothe core component. The at least one ligand comprises at least one of anactive agent, a targeting agent, an imaging agent, a hydrophilicdispersing agent, and combinations thereof. The at least one ligand iseffective to reduce luminescence of the quantum dot when linked thereto.

In accordance with another aspect, there is provided an opticallyactivatable nanoprobe for monitoring intracellular drug delivery. Theoptically activatable nanoprobe comprises an inorganic core and aplurality of quantum dots linked to the inorganic core. A plurality ofligands are linked to respective ones of the plurality of quantum dots.The plurality of ligands include each of a biologically active agent, atargeting agent, and a hydrophilic dispersing agent. Since the ligandsare electron rich, the ligands collectively reduce luminescence of theof the quantum dot when linked thereto.

In accordance with another aspect, there is provided an opticallyactivatable nanoprobe comprising a quantum dot core and at least oneligand linked to the quantum dot core. A chitosan-based shell surroundsthe quantum dot core and the at least one ligand.

In accordance with another aspect, there is provided a method formonitoring intracellular drug delivery within a subject. The methodcomprises administering to the subject an effective amount of anoptically activatable nanoprobe. The optically activatable nanoprobecomprises an inorganic core, a plurality of quantum dots linked to theinorganic core, and at least an active agent linked to the respectiveones of the quantum dots. Upon intracellular uptake of the nanoprobe, alinkage between the active agents and the quantum dots is cleaved toallow the plurality of quantum dots to transfer from a quenched state,wherein the luminescence of the plurality of quantum dots is quenched,to a luminescent state, wherein the luminescence of the plurality ofquantum dot is activated. In one embodiment, the method furthercomprises detecting a presence of the plurality of quantum dots, whereinan increase in luminescence of the plurality of quantum dots isindicative of a release of the active agent intracellularly. In oneembodiment, the at least one ligand comprises each of an active agent, atargeting agent, an imaging agent, and a hydrophilic dispersing agent,or a combination thereof.

In yet another aspect, there is provided a method for monitoringintracellular drug delivery within a subject. The method comprisesadministering to the subject an effective amount of an opticallyactivatable nanoprobe. The optically activatable nanoprobe comprises acore component comprising a quantum dot and an active agent linked tothe quantum dot. Upon intracellular uptake of the nanoprobe, a linkagebetween the active agent and the core component is cleaved to allow forrelease of the active agent and to allow the quantum dot to transferfrom a quenched state, wherein the luminescence of the plurality ofquantum dot is quenched, to a luminescent state, wherein theluminescence of the plurality of quantum dots is activated. The methodfurther comprises detecting a presence of the quantum dot, wherein anincrease in luminescence of the quantum dot is indicative of a releaseof the active agent intracellularly.

In yet another aspect, there is provided an activatable nanoprobecomprising a core component; an activatable active agent associated withthe core component; and a lipid vesicle at least partially or fullyencasing the activatable agent and the core component.

In still another aspect, there is provided an optically activatablenanoprobe comprising: an inorganic core; a plurality of quantum dotsabout the inorganic core and linked to the inorganic core; and ahydrophilic dispersing agent linked to respective ones of a plurality ofthe quantum dots.

In still another aspect, there is provided a method for making opticallyactivatable nanoprobes. The method comprises obtaining a plurality ofnanoparticles comprising an inorganic core and obtaining a plurality ofquantum dots. Further, the method comprises linking the plurality ofquantum dots to the inorganic core; and linking an active agent, atargeting agent, and a hydrophilic dispersing agent to respective onesof the plurality of quantum dots.

In yet another aspect, there is provided a method for monitoringintracellular drug delivery within a subject in whom an effective amountof an optically activatable nanoprobe has been administered. Theoptically activatable nanoprobe comprises an inorganic core; a pluralityof quantum dots linked to the inorganic core, and at least one ligandlinked to respective ones of the plurality of quantum dots, the at leastone ligand comprising at least an active agent linked to the quantum dotby a linkage; and wherein, upon intracellular uptake of the nanoprobe, alinkage between the active agent and a respective quantum dot is cleavedto allow for release of the active agent and to allow the plurality ofquantum dots to transfer from a quenched state, wherein the luminescenceof the plurality of quantum dots is quenched, to a luminescent state,and wherein the luminescence of the plurality of quantum dot isactivated. The method further comprises confirming release of thebiologically active agent by detecting a presence of the plurality ofquantum dots in the luminescent state.

In yet another aspect, there is provided an optically activatablenanoprobe comprising a core component comprising a quantum dot; and acoating comprising a hydrophilic dispersing agent at least partiallysurrounding the core component.

In certain aspects, the nanoprobes include a core component. In oneembodiment, the core component comprises an inorganic core, which allowsfor the attachment of a plurality of a Qdots to be disposed about theinorganic core. In this way, a single activatable nanoprobe may have aplurality of quantum dots attached thereto for signal enhancement inmonitoring applications. In one embodiment, the inorganic core comprisesa paramagnetic material. In a particular embodiment, the inorganic corecomprises iron oxide. A super-paramagnetic iron oxide nanoparticle is anexcellent MRI contrast agent (also called a T2 contrast agent).Utilizing iron oxide in the inorganic core facilitates imaging of MMCNPsby MRI. It also assists in purification, as an external permanent magnetcan be implemented to separate MMCNPs magnetically from the reactionmixture.

The Qdots may comprise a semiconductor crystal whose size is on theorder of just a few nanometers and that exhibits quantum confinement.Typically, Qdots contain anywhere from about 100 to about 1,000essentially free electrons and range from about 2 nm to about 10 nm insize, or about 10 to about 50 atoms, in diameter. One of the opticalfeatures of an excitonic Qdot noticeable to the unaided eye iscoloration. While the material which makes up a quantum dot issignificant, more significant in terms of coloration is the size. Thelarger the Qdot, the redder (the more towards the longer wavelength endof the electromagnetic spectrum) they fluoresce. The smaller the Qdot,the bluer (the more towards the short wavelength end) it is. Thecoloration is directly related to the energy levels of the Qdot.Quantitatively speaking, the band gap energy that determines the energy(and hence color) of the fluoresced light is approximately inverselyproportional to the square of the size of the Qdot. Larger Qdots havemore energy levels, which are more closely spaced. This allows the Qdotto absorb photons containing less energy, e.g., those closer to the redend of the spectrum.

In certain embodiments, the Qdots include a semiconductor nanocrystalcore and a wide band-gap semiconductor nanocrystalline shell (coating)disposed on the surface of the nanocrystal core, which is typicallydifferent from the material used for the nanocrystal core. Thesemiconductor coating may define a fully or partially passivatingsemiconductor coating layer. Exemplary materials for the nanocrystalcore and the semiconducting coating of the quantum dot include, but arenot limited to, zinc sulfide, zinc selenide, zinc telluride, cadmiumsulfide, cadmium selenide, cadmium telluride, mercury sulfide, mercuryselenide, mercury telluride, magnesium telluride, aluminum phosphide,aluminum arsenide, aluminum antimonide, gallium nitride, galliumphosphide, gallium arsenide, gallium antimonide, indium nitride, indiumphosphide, indium arsenide, indium antimonide, aluminum sulfide, leadsulfide, lead selenide, germanium, silicon, other group II-group VIcompounds, group III-group V compounds, group IV compounds, and alloys,compounds, or mixtures thereof. In one embodiment, the semiconductorcoating has a band gap greater than the band gap of the nanocrystalcore. In some embodiments, the core material can also be doped with oneor more suitable dopants, such as manganese (Mn) or copper (Cu). In theexamples below, a nanoprobe comprising an iron oxide nanoparticle coreassociated with satellite CdS:Mn/ZnS Qdots was formed, although it isunderstood the present invention is not so limited. In anotherembodiment, the Qdots may comprise ZnS:Mn/ZnS Qdots (non-heavy metalcontaining Qdots such as cadmium or arsenic).

In particular embodiments, a coupling agent may be provided to link theQdots to the inorganic core. In one embodiment, the coupling agentcomprises a heterobifunctional cross-linking compound that can link tothe inorganic core at one location thereon and can link to a respectiveQdot at a location thereon. In one embodiment, the coupling agentcomprises dihydrolipoic acid (DHLA), which can link the inorganic corethereto through its carboxyl end and link to a respective Qdot via itsbidentate thiol bonds.

Either or both of the inorganic core or the quantum dots can be modifiedwith the coupling agent prior to addition of the other of thecomponents. In one embodiment, the coupling agent is provided as acoating over the inorganic core. As set forth below, in one embodiment,to minimize the possibility of cross-linking when using a couplingagent, e.g., DHLA, the addition of the modified inorganic core particlesto unmodified quantum dots may be done slowly and in a controlledmanner.

When the ligand comprises a targeting agent, the targeting agent may beany compound having an affinity for a predetermined molecular target. Inone embodiment, the targeting agent comprises a folate species, whichmay be folate, folic acid, or derivatives thereof. Examples of folatederivatives include, but are not limited to, dihydrofolate,tetrahydrofolate, 5,-methyl-tetrahydrofolate and 5,10-methylenetetrahydrofolate. Humans and other mammals express a number of proteinsthat bind to folate and transport it into cells. For example, in humans,alpha and beta folate receptors have been identified, each of which canoccur in several isoforms (e.g. as a result of differentialglycosylation). These proteins are referred to as “folate receptors.”Thus, a folate receptor is considered to be any protein expressed on thesurface of a cell, such as a cancer cell, which binds folate inpreference to other moieties or compounds. In one embodiment, thetargeting agent is selected to specifically target these folatereceptors in a mammalian subject.

Additionally, in other embodiments, the targeting agent may be one ormore of an aptamer, a peptide, an oligonucleotide, an antigen, anantibody, or combinations thereof having an affinity for a predeterminedmolecular target. In one embodiment, the predetermined molecular targetis associated with a cancer cell, a leukemia cell, an acutelymphoblastic leukemia T-cell, or combinations thereof. In a particularembodiment, the ligand comprises an aptamer having an affinity forleukemia cells, e.g., an acute lymphoblastic leukemia T-cell. Theaptamer may include any polynucleotide- or peptide-based molecule, forexample. A polynucleotidal aptamer is a DNA or RNA molecule, usuallycomprising several strands of nucleic acids that adopt highly specificthree-dimensional conformation designed to have appropriate bindingaffinities and specificities towards specific target molecules, such aspeptides, proteins, drugs, vitamins, among other organic and inorganicmolecules. Such polynucleotidal aptamers can be selected from a vastpopulation of random sequences through the use of systematic evolutionof ligands by exponential enrichment. A peptide aptamer is typically aloop of about 10 to about 20 amino acids attached to a protein scaffoldthat bind to specific ligands. Peptide aptamers may be identified andisolated from combinatorial libraries, using methods such as the yeasttwo-hybrid system. In one embodiment, the ligand comprises the DNAaptamer sgc8c having a sequence according to SEQ. ID No. 1:

5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3′.

The DNA aptamer sgc8c has been shown to have a particular bindingaffinity for leukemia cells, e.g., acute lymphoblastic leukemia T-cells.

When the ligand comprises a hydrophilic dispersing agent, thehydrophilic dispersing agent may be directly or indirectly linked torespective ones of the quantum dots. In addition, when the ligandcomprises a hydrophilic dispersing agent, the hydrophilic dispersingagent may comprise any compound that will increase a hydrophilicity ofthe nanoprobe relative to a nanoprobe without the hydrophilic dispersingagent. Exemplary hydrophilic dispersing agents include but are notlimited to one or more of NAC (N-Acetyl-L-Cysteine (NAC), glutathione(GSH), PEG (polyethylene glycol), m-PEG, PPG (polypropylene glycol),m-PPG, PGA (polyglutamic acid), polysialic acid, polyaspartate,polylysine, polyethyeleneimine, biodegradable polymers (e.g.,polylactide, polyglyceride), and functionalized PEG, e.g.,terminal-functionalized PEG, and structural analogues of any of theabove compounds.

In one embodiment, the hydrophilic dispersing agent is indirectly linkedto respective ones of the quantum dots through a spacer or linkingcompound. For example, as will be explained below, in a particularembodiment, a NAC(N-Acetyl-L-Cysteine)-modified ethylenediamine ligand(NAC-EDA modified ligand) is first linked to the quantum dot surface viathe sulfide groups of NAC. Thereafter, the surface amine groups providedby NAC-EDA may be reacted with an N-hydroxysuccinimide (NHS) esterderivative of methyl-poly-ethylene glycol (MPEG-NHS ester) to improveoverall dispersability of the nanoprobe.

In a particular embodiment, a plurality of hydrophilic dispersing agentsare linked to a plurality of the Qdots so as to define a hydrophilicshell or coating for the nanoprobe. In one embodiment, a hydrophilicshell or coating is formed by linking a plurality of hydrophilicdispersing molecules to a plurality of Qdots surrounding the inorganiccore. It is appreciated that other ligands may be attached to the Qdotsas described herein, e.g., an active agent or a target agent, and thatthe hydrophilic shell or coating may not be continuous. Typically, thecoating or shell will at least partially surround the inorganic core. Inone embodiment, the hydrophilic dispersing agent comprises GSH. Inanother embodiment, the hydrophilic dispersing agent comprises NAC. Asset forth in the Example, the present inventors have found that GSH andNAC are at least capable of coating a Qdot surface via conjugationthrough their sulfhydryl (—SH) groups, and thus form “hydrophilic Qdots”or “hydrophobic nanoprobes.” Both GSH-Qdots and NAC-Qdots, andparticularly NAC-Qdots, may be an attractive choice for the fabricationof activatable (“OFF/ON”) Qdots for bioimaging and sensing applications.

When the ligand of the optically activatable nanoprobe comprises anactive agent, the active agent may include any compound or compositionthat produces a preventative, healing, curative, stabilizing,ameliorative or other beneficial therapeutic effect. In a particularembodiment, the active agent is a STAT inhibitor, such as a STAT-3inhibitor. Examples of mammalian STAT inhibitors include inhibitors ofSTAT-1, STAT-2, STAT-3, STAT-4, STAT-5a, STAT-5b, and STAT-6. In oneembodiment, the active agent is one or more of BP-1-102, SF-1066 andS31-201, all of which have proven activity against STAT-3 with IC₅₀values of 6.8, 35, and 86 μm, respectively.

STAT-3, in particular, is an oncogene constitutively activated in manycancer systems where it contributes to carcinogenesis. The signaltransducers and activators of transcription (STATs) are a class oftranscription factor proteins that regulate cell growth and survival. Atotal of seven STAT isoforms, encoded in distinct genes, have beenidentified in mammalian cells. STAT-3 protein isoform is known todirectly up-regulate Bcl-xL, Mcl-1, cyclin D1/D2 and c-myc, contributingto compromised regulation by stimulating cell proliferation andpreventing apoptosis in numerous human cancers including breast,prostate, melanoma, lung, brain, pancreatic, ovarian, colon cancers.STAT-3 activation occurs via phosphorylation of tyrosine 705, whichpromotes STAT dimer formation through STAT phosphotyrosine-SH2 domaininteractions. These STAT dimers translocate to the nucleus, where theyregulate gene expression. Constitutive STAT-3 activity mediatesdysregulated growth and survival, angiogenesis, as well as suppressesthe host's immune surveillance of tumors and represents a valid targetfor small molecule anti-cancer design. The STAT-3 inhibitor for use inthe present invention may be any known inhibitor of STAT3 known in theart. Exemplary STAT3 inhibitors are disclosed in U.S. Published PatentApplication Nos. 20100310645, 20090318367, 20090069420, 20080187992,20070123502, 20050074502, and 20050004009, the entirety each of which isincorporated by reference herein. Typically, the STAT-3 inhibitor willinclude a sulfur-containing functional group, which may form a covalentdisulfide bond with the Qdot to form a linkage between the STAT-3inhibitor and the Qdot.

The optically activatable nanoprobes described herein may thus beutilized to monitor and/or treat any proliferation disordercharacterized by the over-activation of a STAT protein. In oneembodiment, the proliferation disorder to be treated is a cancerproducing a tumor characterized by over-activation of STAT1, STAT3,STATS, or a combination of two or all three of the foregoing. Examplesof such cancer types include, but are not limited to, breast cancer,ovarian cancer, multiple myeloma and blood malignancies, such as acutemyelogenous leukemia. In addition to cancer, the proliferation disorderto be treated using the compounds, compositions, and methods of theinvention can be one characterized by aberrant STAT-3 activation withincells associated with a non-malignant disease, pathological state ordisorder (collectively “disease”), and likewise comprising administeringor contacting the cells with a an effective amount of one or more STAT3inhibitors to reduce or inhibit the proliferation.

In still other embodiments, the active agent may include peptides (e.g.,RGD peptide, integrin selective; see Dechantsreiter, M. A., et al.,N-Methylated Cyclic RGD Peptides as Highly Active and Selective αvβ3Integrin Antagonists. Journal of Medicinal Chemistry, 1999. 42(16): p.3033-3040.), antibodies (e.g., CD10 monoclonal antibody for targetinghuman leukemia; see Santra, S., et al., Conjugation of Biomolecules withLuminophore-Doped Silica Nanoparticles for Photostable Biomarkers.Analytical Chemistry, 2001. 73(20): p. 4988-4993.) and proteins. Whenthe optically activatable nanoprobes comprise a biologically activedrug, as well a target-specific ligand, the disclosed nanoprobes areuseful as target-specific drug delivery vehicles.

The imaging agent may comprise one or more of a fluorophore, iohexyl,and a paramagnetic chelate having a paramagnetic ion bound therein. Inanother embodiment, the imaging agent may be a fluorophore and/or aparamagnetic chelate (chelator) having an MRI (magnetic resonanceimaging) contrast agent bound therein. The MRI contrast agent maycomprise a paramagnetic ion selected from one or more of gadolinium,dysprosium, europium, and compounds, or combinations thereof, forexample. In one embodiment, the paramagnetic ion comprises a gadoliniumion and the chelator is a DOTA-NHS ester(2,2′,2″-(10-(2-(2.5-dioxopyrrolidin-1yloxy)-2-oxoethyl)-14,7,10-tetraazacyclododecane-1,4,7-tryl)triaceticacid).Gd³⁺ ions are paramagnetic and DOTA is a chelator of Gd ion. The Gd-DOTAis paramagnetic agent and it provides MRI contrast. Gd-DOTA iscommercially available under the brand name ProHance® (also calledGadoteridol). In another embodiment, the imaging agent may compriseiohexyl.

In accordance with one aspect of the invention, the ligand(s) (e.g., atargeting agent, dispersing agent, and/or an active agent), thecross-linker or spacing compound, and/or the Qdot may be modified with acompound that increases the functionality and/or dispersability of theoptically activatable nanoprobe in aqueous solutions, particularly at pHof 7.4. In one embodiment, the ligand(s) (e.g., a targeting agent,dispersing agent, and/or an active agent), the linking or spacingcompound, and/or the quantum dot may be modified with NAC(N-Acetyl-L-Cysteine). NAC-modification of the Qdot, ligand, orlinking/spacing molecules has several advantages: (i) NAC passivates theQdot surface via formation of stable disulfide bonds, resulting inincreased quantum efficiency; ii) NAC improves aqueous dispersability ofthe nanoprobes; and iii) NAC provides surface carboxyl groups forfunctionalization with other desired ligands.

The below description further describes exemplary embodiments of anoptically activatable nanoprobe in accordance with the present inventionand a method for making the same. It is understood that the presentinvention, however, is not limited to the below-described opticallyactivatable nanoprobes, and that the nanoprobes may include one, two, ormore of the ligands described below attached to the Qdots, may includeother linked compounds, and may be further modified as necessary for theparticular application.

In one embodiment, as shown in FIG. 1a , the optically activatablenanoprobe (MMCNP) comprises a super-paramagnetic iron oxide nanoparticlecore (IONP; ˜5-20 nm size) and satellite CdS:Mn/ZnS quantum dot (Qdots;˜3.5 nm size) shell. Each Qdot is attached to the core IONP by ahetero-bifunctional cross-linker molecule, dihydrolipoic acid (DHLA).The DHLA connects the IONP through its carboxyl end and to the Qdot viaits bidentate thiol bonds. To minimize the possibility of cross-linking,the IONP-Qdot conjugation strategy involved controlled addition of DHLAmodified IONP to unmodified Qdots as described in the Example below. Thecarboxyl and the thiol functional groups are compatible with the IONPand Qdot particle surfaces, respectively^(18,24-26).

Upon attachment of a Qdots to an IONP nanocrystal, a large surface areastill remains available on the satellite Qdots for further surfacemodification and conjugation. Next, an N-Acetyl-L-Cysteine(NAC)-modified STAT3 inhibitor (NAC-STAT3; a therapeutic model drug), aNAC-modified folate (NAC-FA; a cancer targeting agent) and anNAC-modified ethylenediamine (NAC-EDA, an amine modified ligand) wereseparately synthesized. As set forth above, NAC-mediated surfacemodification of Qdots has several advantages: (i) it passivates the Qdotsurface via formation of stable disulfide bonds, resulting in increasedquantum efficiency, (ii) it improves aqueous dispersibility of Qdots,and (iii) it provides surface carboxyl groups for functionalization withother desired ligands. Furthermore, this approach reflects theuniqueness of the reported MMCNP design, where the separate synthesis ofeach of these ligands allows for control of the ratio of these ligandswhen attaching to Qdots or substitution of one of these ligands,resulting in a fully modular design of the MMCNP (akin to a nanoparticleLEGO®). After IONP-Qdot conjugation, further surface conjugationreactions were performed by treating IONP-Qdot with a mixture ofNAC-STAT3, NAC-FA and NAC-EDA. The surface modification procedures aredetailed in Examples below. The surface amine groups (provided byNAC-EDA) were reacted with the N-hydroxysuccinimide (NHS) esterderivative of methyl-poly-ethylene glycol (MPEG-NHS ester; abiocompatible highly-hydrophilic dispersing agent) to improve overalldispersibility of the MMCNPs.

A STAT-3 drug and folate (FA) were intentionally selected aselectron-rich ligands that can substantially quench Qdot fluorescence.Substantial quenching typically refers to 75-100% quenching offluorescence. In a more specific embodiment, substantial quenchingrefers to 90-100% fluorescence quenching. This selection processinvolved mixing of each of these ligands with Qdots followed byobservation of the extent of luminescence quenching (data not shown).Each of these ligands thus serves a dual purpose. The treatment ofIONP-Qdots with NAC-STAT3 and NAC-FA drastically reduced thefluorescence of the Qdots. It was noted that NAC itself did not quenchQdot luminescence, thus justifying the combined role of STAT-3 drug andFA as quenchers. As a result, the MMCNP is essentially in afluorescently quenched (“OFF”) state when the ligand(s) are attached tothe Qdots. It is understood that by “OFF,” however, it is notnecessarily meant the nanoprobe is 100% quenched or does not exhibitsome luminescence, but only that the amount of luminescence is greaterwhen the ligand(s) are not attached vs. attached. The luminescence ofthe MMCNPs is restored (“ON” state) upon treatment with an appropriatecleaving agent, which effectively cleaves disulfide bonds between theligand and the IONP-Qdots, such as glutathione (GSH).

The morphological, optical, and magnetic properties of MMNCPs wereextensively characterized. TEM studies confirm the formation of about20-40 nm size nanocomposites as shown in FIG. 1b . The IONP core andsatellite Qdots surrounding the core are clearly visible with lowresolution TEM (FIG. 1b ), whereas magnified TEM images clearly showQdots around the IONP (FIG. 1b inset). HRTEM also confirms the singlecrystalline structure of the Qdots (FIG. 7) surrounding the core IONP.Inductively coupled plasma analysis of the sample confirms the presenceof Zn (about 41 wt %), Fe (about 6.2 wt %) and Cd (about 10 wt %) with arelative ratio of about 6.6:1.0:1.6 (W/W). Zeta potential (ξ)measurements correlate with particle surface charge. The ξ values ofIONP, IONP-DHLA, IONP-Qdots-STAT3-FA and IONP-Qdot-STAT3-FA-m PEG areabout −20 mV, about −4.0 mV, about −17 mV and about −21 mV,respectively. As expected, DHLA modification of the iron oxidenanoparticle core (IONP) drastically reduced its surface charge due toreduction of the negative surface charge of the IONP. Further surfacemodification with the STAT-3 drug and folic acid (folate) resulted in anincrease in the overall negative surface charge on the particle. This islikely due to the presence of carboxyl groups on the folate residues onthe particle surface.

Pegylation with biocompatible mPEG, however, showed minimal effect onthe particle surface charge due to its neutral nature. It was observedthat pegylated particles exhibited good phosphate buffer dispersibility.A comparative analysis of FT-IR spectra of dihydrolipoic acid (DHLA)(a), lipoic acid (b), DHLA-coated IONPs (c), and IONPs (d) confirmedsuccessful surface modification of IONP with DHLA (FIG. 8). Fluorescencespectroscopy in solution was used to investigate the Qdot luminscenceproperties at different stages of MMCNP development as well as fortracking the drug release events.

Glutathione (GSH), a tripeptide biomolecule found in all animal cells atrelatively high cytosolic concentration (about 1-10 mM^(27,28), reducedform), effectively reduces disulfide bonds and in this processglutathione is converted to glutathione disulfide (GSSG), its oxidizedform.²⁹ The design of the MMCNPs is such that once they are exposed toan intracellular GSH environment, the nanoprobes will break apart intoits different constituents that make up the composite nanoprobes. Thisforms the basis of the intracellular tracking of the STAT-3 drug releaseas schematically shown in FIG. 1a . Distinct changes in absorptionspectra were observed for IONP-Qdot and IONP-Qdot-STAT3 conjugates incomparison to Qdots (FIG. 9). The Qdot absorption spectrum broadenssignificantly and slightly shifts towards longer wavelength whenconjugated to IONPs. However, upon further conjugation with NAC-STAT3drug, NAC-FA and NAC-EDA, a decrease in spectral width along with slightblue shift was observed with respect to the IONP-Qdots conjugates. Suchchanges in absorption spectral characteristics support successfulsurface conjugation of Qdots with IONP, STAT3 drug and FA. The emissionof MMCNP is slightly blue shifted compared to either Qdots or IONP-Qdotconjugates (FIG. 10).

Fluorescence data acquired by adding GSH to MMCNPs in solution show thatQdot fluorescence can be substantially restored in less than one hour(FIGS. 2a and 2b ). Furthermore, a systematic study on the effect of GSHconcentration on the time scale of fluorescence restoration shows thatthere is no significant effect of varying GSH concentrations in therange of 2.8 mM to 7 mM (FIG. 11). The observed spectral features of the“ON” state Qdots (i.e. after release from MMCNP) (FIG. 2a ) are in goodagreement with those of Qdots in solution as shown in FIG. 10. TheSTAT-3 inhibitor is also a fluorescent molecule (λ_(ex): 300 nm andλ_(em): 396 nm) of which the fluorescence is substantially quenched inMMCNPs. Fluorescence quenching of STAT-3 drug is presumably due toelectron/energy transfer from STAT-3 to Qdots. It is unlikely that theIONP will have any significant contribution towards STAT-3 fluorescencequenching as STAT-3 is not directly attached to IONP surface.Restoration of STAT-3 drug emission is observed once MMCNPs are treatedwith GSH in solution (FIG. 2b ), thus confirming disintegration ofMMCNPs and release of STAT-3 inhibitors.

To confirm the effectiveness of the optically activatable nanoprobes(MMCNPs), the MMCNPs were challenged against the intracellular GSHenvironment where the reported GSH concentration is in the millimolar(mM) range, typically between about 2 mM and about 15 mM^(27,28). Thehuman breast cancer (MDA-MB-231) cell line, known to over-express folatereceptors³⁰, and the mouse thymus stromal epithelial cell line (TE-71)were incubated for up to 24 hrs with MMCNP at a concentration of about50 μg/mL. As expected, a significant uptake of folate conjugated MMCNPsby the cancer cells was observed compared to normal cells as shown inFIG. 3. These results also show that substantial restoration offluorescence occurs within 3 hr incubation. Restoration of fluorescencein cancer cells is a direct confirmation of targeted cellular uptake ofMMCNPs and subsequent dispersal of MMCNPs into its separate components,e.g., IONP, Qdots, and the release of ligands, including drug molecules.

Systematic optical studies were performed to investigate intracellulardrug release at the single cell level. Confocal microscopy images of theMDA-MB-231 cells incubated with MMCNPs for 5 hrs (FIGS. 4a-4c ) clearlyshow that MMCNPs were uptaken by the cells. In addition, strongfluorescence signal from only a few locations in the cell can beobserved. These data show that Qdots were released from the MMCNPthrough the cleavage of disulfide bonds by GSH (see FIG. 1a ). Besidesthe images of single cells incubated with MMCNPs, emission spectra ofdifferent regions in single cells were also collected (FIG. 4c , red andcyan lines). The Qdots in the intracellular environment show emissionspectra that are red-shifted and broadened with respect to uncoated freeQdots in solution (FIG. 10). These spectral differences are attributedto aggregation of the Qdots after release from MMCNP in the cytosol.This observation was confirmed with solution experiments on bare Qdotsby observing emission spectra before and after aggregation, as well asaddition of GSH to each of these samples (data not shown).

It was found from control experiments that addition of GSH to asuspension of bare Qdots leads to a stable Qdot suspension and has nonoticeable effect on the Qdot emission properties (data not shown).These observations may provide preliminary indication that in theintracellular environment GSH does not necessarily exchange with theNAC/cargo-ligand that is initially present on the Qdot surface due tothe fact that intracellular Qdot aggregation is observed after cargorelease, although it could be argued that binding constants of bothmolecules could be comparable given that GSH and NAC both contain asingle thiol group in their structure (monodentate ligand).

The observation of only a few bright spots in a single cell in thefluorescence images indicates aggregation of multiple Qdots in a singleor a few clusters. The Qdot aggregation is reasonable given that whilein the MMCNP, Qdots are stabilized by PEG. After exposure to GSH, thePEG is removed by cleavage of disulfide bonds, resulting in hydrophobicQdots that self-aggregate. In addition, the data shows that these Qdotaggregates preferentially localize near the cell membrane, again due totheir hydrophobic nature. Other locations where Qdot aggregates are notpresent only show autofluorescence. It should be noted that while theSTAT3 drug itself is typically also fluorescent (FIG. 2b ), experimentson intracellular delivery cannot be reliably performed by measuring theSTAT3 drug fluorescence due to weak fluorescence and the presence ofcellular auto-fluorescence, hence the need for the optical signal of theQdots.

To demonstrate the concept of multimodality of the MMCNPs, an agarphantom was prepared using a 10 mm NMR tube (FIGS. 5a-5d ) for MRI andoptical imaging studies. FIG. 5a shows the schematic of agar phantomdesign. The bottom part of the tube contains only MDA-MB-231 cells (as acontrol), middle part contains only MMCNPs (as a control) and the toppart of the tube contains the same cells loaded with MMCNPs. All ofthese were dispersed in 3% agarose gel under same condition. A digitalimage of the tube was recorded under room light (FIG. 5b ) as well asunder illumination by a hand-held 366 nm multiband UV lamp (FIG. 5c ).The unfiltered images clearly show a light brown color where cells arelocated in room light conditions whereas an intense red color appearsdue to Qdot fluorescence under UV illumination. The MDA-MB-231 cellsloaded with the MMCNPs emit red fluorescence that is clearly visible tothe naked eye. Control cells do not show any detectable fluorescenceemission. An MRI image of the phantom (FIG. 5d ) clearly shows cellclusters that correlate well with the fluorescence image. A 3Dreconstruction of the MR images is provided in FIG. 13. Thisdemonstrated the appearance of strong MRI signal from the MDA-MB-231cells loaded with the MMCNPs (indicated with false red color) incontrast with the control cells.

A CyQUANT™ cell proliferation assay was used in a comparative cellviability study to determine cytotoxicity of MMCNPs without STAT-3 drug(control particle), STAT-3 drug itself, and MMCNPs with STAT-3conjugation. Two cancer cell lines, MDA-MB-231 and pancreatic (Panc-1)cancer cells were used along with mouse thymus stromal epithelial TE-71cells (control). Results (shown in FIG. 6) suggest that MMCNPs (withSTAT-3) treated cancer cells have lower viability than cells treatedwith free STAT-3 drug when MMCNPs and STAT-3 were administered to thecell medium at identical concentrations. This finding is highlysignificant given that free STAT3 drug alone in medium is in high excesscompared to STAT-3 drug present in MMCNPs under these conditions. Eventhough the MMCNPs consume much less STAT3 drug, the delivery efficiencyis dramatically increased, thus resulting in increased therapeuticefficiency while minimizing the potential for medical side effects dueto presence of excess free drug.

In view of the above, a new and unique quantum dot (Qdot)-iron oxide(10) based multimodal/multifunctional nanocomposite probe that isoptically and magnetically imageable, targetable and capable ofreporting on intracellular drug release events has been describedherein. By design, the present nanoparticle system has multimodalities(optically and magnetically active) and multifunctionalities (i.e.imaging, targeting, drug delivery) that are current state-of-the-art innanomedicine research^(1,8,10,14-16,18,23-26). In addition, the MMCNPdiscussed here integrate sensing modalities that report on the event aswell as the location of intra-cellular release of cargo (drug, etc.).The impact and implications of this new development along with thetraditional multimodalities and multifunctionalities are immediate fordrug discovery and cancer biology.

In accordance with another aspect, there is provided a method formonitoring intracellular drug delivery within a subject using anyembodiment of an optically activatable nanoprobe described herein. Inone embodiment, the method comprises administering to the subject aneffective amount of a nanoprobe comprising an inorganic core and aplurality of quantum dots linked to the inorganic core. A ligandcomprising at least an active agent is linked to respective ones of theplurality of quantum dots. Upon intracellular uptake of the nanoprobe,the linkage between the active agent and the ligand is cleaved to allowrelease of the active agent and to allow the plurality of quantum dotsto transfer from a quenched state, wherein the luminescence of theplurality of quantum dots is quenched, to a luminescent state, whereinthe luminescence of the plurality of quantum dot is activated. In oneembodiment, the linkage between the bioactive agent and the quantum dotis cleavable by intracellular glutathione (GSH).

In a particular embodiment, the ligand may further comprise a targetingagent and a hydrophilic dispersing agent. For example, the active agentmay comprise a STAT3 inhibitor, the targeting agent may comprise folate,and the hydrophilic dispersing agent may comprise m-polyethylene glycol(mPEG) or derivatives thereof. In addition, optionally, the bioactiveagent, targeting agent, and the hydrophilic dispersing agent may bemodified with N-Acetyl-L-Cysteine to provide improved dispersability ofthe nanoprobes and additional functional groups for the attachment ofdesired ligands.

In addition, the above-described method may further comprise the step ofdetecting a presence of the plurality of quantum dots, wherein anincrease in luminescence of the quantum dots is indicative of a releaseof the active agent intracellularly. The detecting may be done by anysuitable detection method known in the art appropriate for theparticular type of Qdot. For example, the detection may be done by anyone or more of transmission electron microscopy (TEM), scanning electronmicroscopy (SEM), dynamic light scattering (DLS), UV-visible (UV-VIS)spectroscopy, Fourier transform infrared spectroscopy (FTIR), zetapotential, high pressure liquid chromatography-mass spectrometry(HPLC-MS), NMR/IR, mass spectrometry (MS), fluorescence excitation andemission spectroscopy and fluorescence microscopy, near infrared (NIR)imaging (NIRS), and magnetic resonance (MR) imaging and spectroscopy

The administering to the subject, who may be any mammalian subject, maybe done according to any suitable route of in vivo administration thatis suitable for delivering the composition into the subject. Thepreferred routes of administration will be apparent to those of skill inthe art, depending on the medium, the targeting agent (if present) orthe active agent. Exemplary methods of in vivo administration include,but are not limited to, intravenous administration, intraperitonealadministration, intramuscular administration, intranodal administration,intracoronary administration, intraarterial administration (e.g., into acarotid artery), subcutaneous administration, transdermal delivery,intratracheal administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, intranasal, oral, bronchial,rectal, topical, vaginal, urethral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue.

In accordance with another aspect, there is provided a method for makingoptically activatable nanoprobes as described herein. The methodcomprises obtaining a plurality of nanoparticles comprising an inorganiccore, e.g., iron oxide core; obtaining a plurality of quantum dots;linking the plurality of quantum dots to the inorganic core; and linkingat least one ligand to respective ones of the plurality of quantum dots,wherein the at least one ligand comprises an active agent, a targetingagent, or a hydrophilic dispersing agent. In a particular embodiment,the at least one ligand comprises an active agent, a targeting agent,and a hydrophilic dispersing agent. As discussed above, the active agentmay comprise a STAT-3 inhibitor, the targeting agent comprises folate,and the hydrophilic dispersing agent comprises polyethylene glycol(e.g., m PEG).

Further, in one embodiment, the method further comprises providing acoating of dihydrolipoic acid (DHLA) about the inorganic core toconnect/link the Qdots to the iron oxide core. In some embodiments, themethod further comprises modifying at least one of the active agent, thetargeting agent, and the hydrophilic dispersing agent withN-Acetyl-L-Cysteine to increase dispersability and multifunctionality ofthe nanoprobe. A specific process for making optically activatablenanoprobes as described herein is set forth in the example below.

In accordance with another aspect of the present invention, there isprovided another embodiment of a multi-modal, multi-functionalnanoparticle (hereinafter DNCP or DNCPs) that allows for enhanced invivo efficacy of active agents, as well as allows for the improvednon-invasive in vivo bio-imaging. In one embodiment, the DNCPs comprisean optically activatable nanoprobe having a core component. In thisembodiment, the core component comprises a quantum dot core and at leastone ligand linked to the quantum dot core. In this embodiment, the DNCPsdiffer from the above-described nanoparticles in that the Qdot itselfcomprises the nanoparticles' core rather in contrast to the inorganiccore described above having a plurality of satellite Qdots surround aninorganic core. A number of different chemical entities may be linked tothe DNCPs, such as active agents, imaging agents, and targeting agentsaccording to the same structures and methods described above.

In one embodiment, at least one active agent, e.g., a NAC-modified STAT3inhibitor, is linked to the Qdot core by a disulfide bond, for example.In this way, when the STAT3 inhibitor is linked to the Qdot core, theSTAT3 drug attachment to the Qdot surface will substantially quench Qdotfluorescence. The restoration of Qdot fluorescence will take place whenintracellular GSH acts upon the DCNP, cleaving the disulfide bond andreleasing STAT3 drug from the DNCP core into the cytosol. Restoration offluorescence typically involves at least a noticeable increase offluorescence from a substantially quenched state.

In one embodiment, a chitosan-based shell surrounds the Qdot core andany chemical entities attached thereto in the DCNPs. In one embodiment,the chitosan-based shell comprises a chitosan polymer and a hydrophilicdispersing agent. Chains of the chitosan polymer electrostaticallyinteract with chains of the hydrophilic dispersing agent to form anentangled network comprising the chitosan polymer and the hydrophilicdispersing agent. While not wishing to be bound by theory, it isbelieved that the chitosan-based shell will effectively scavenge ionleakage, e.g., cadmium ion leakage, from the Qdot core by forming ametal-ligand complex and will passivate cytotoxicity. When a pluralityof active agents (e.g., a STAT-3 inhibitor) are attached to the Qdotcore, the plurality of active agents may be released intracellularlyupon cleavage of the bond between the active agents and the Qdot core.

The hydrophilic dispersing agent of the chitosan-based shell may be anycompound having repeating structural units that have one or morefunctional groups that will interact by electrostatic or chargeattraction (or otherwise) with the amine functional groups of thechitosan polymer. In one embodiment, the hydrophilic dispersing agent isa polymer other than chitosan having repeating structural units, whereineach of the structural units includes one or more carboxyl groups. In aparticular embodiment, the hydrophilic dispersing agent comprisespolyglutamic acid (PGA) or any structural analogues or derivativesthereof. The advantages of utilizing PGA as the hydrophilic dispersingagent include the fact that PGA is a negatively charged biocompatibleand biodegradable natural polymer, rendering it suitable for in vivoapplications. Similarly, PGA increases the overall hydrophilicity of thechitosan-based nanoparticles, thus improving the stability of thenanoparticles having PGA therein at physiological pH conditions (e.g.,pH 7.4). Further, PGA provides additional functional groups toincorporate additional functionality and/or modalities to thenanoparticles, such as the attachment of imaging agents, targetingagents, and/or further active agents to the nanoparticles. Even further,the incorporation of PGA in the nanoparticles will reduce the positivesurface charge on each of the nanoparticle's surface (relative to achitosan-based nanoparticle without the PGA), which will likely aid inreducing non-specific uptake by cells.

Alternatively, the hydrophilic dispersing agent may comprise or furthercomprise any other compound that will increase the hydrophilicity of theDNCPs relative to a nanoparticle without the hydrophilic dispersingagent. In other embodiments, for example, the hydrophilic dispersingagent may comprise one or more of NAC, glutathione, PEG (polyethyleneglycol), m-PEG, PPG (polypropylene glycol), m-PPG, polysialic acid,polyaspartate, polylysine, polyethyeleneimine, biodegradable polymers(e.g., polylactide, polyglyceride), and functionalized PEG, e.g.,terminal-functionalized PEG, analogues, derivatives, or combinationsthereof of the above compounds. Optionally, a cross-linking compound maybe provided to conjugate the amine groups of the chitosan polymer andthe carboxyl groups of the hydrophilic dispersing agent together.

The at least one ligand linked to the Qdot may comprise an active agent,a targeting agent, a hydrophilic dispersing agent, an imaging agent, anyother desired compound, and combinations thereof. In one embodiment, thebiologically active agent, targeting agent, and/or imaging agent arelinked to the Qdot core via thiol groups on the Qdot surface. In anotherembodiment, the biologically active agent, targeting agent and/orimaging agent may be linked to the chitosan-based shell, e.g., to eitheror both of the chitosan polymer and the hydrophilic dispersing agent. Itis contemplated that the additional ligands described herein may belinked to the chitosan polymer by bonding, covalent or otherwise,through the amine groups of the chitosan polymer, although the inventionis not so limited. When the chitosan-based shell comprises PGA, therespective ligand may be bonded to the hydrophilic dispersing agentthrough compatible functional groups on the hydrophilic dispersingagent. For example, when the hydrophilic dispersing agent is PGA, theadditional ligand may be bonded to the PGA polymer through its amine orcarboxyl functional groups. In some embodiments, spacer molecules orcoupling agents may be utilized between the ligand to be attached andthe chitosan polymer or the hydrophilic dispersing agent to provide thelinkage between the ligand and the other substrate.

The active agent, imaging agent, hydrophobic dispersing agent, and thetargeting agent may be any compound as described or defined herein.Further, in any embodiment, the active agent, imaging agent, hydrophobicdispersing agent, and the targeting agent may be activatable as defineherein. In one embodiment, the active agent is a STAT3 inhibitor, suchas one or more of BP-1-102, SF-1066 and S31-201, all of which haveproven activity against STAT3 with IC₅₀ values of 6.8, 35, and 86 μm,respectively. FIG. 15 shows the structure of BP-1-102, for example. Asmentioned above, the STAT3 inhibitor may be NAC-modified. FIG. 16illustrates an exemplary NAC-modified STAT3 inhibitor.

The imaging agent may comprise one or more of a fluorophore, iohexyl,and a paramagnetic chelate having a paramagnetic ion bound therein. Inone embodiment, either or both of the hydrophilic dispersing agent andthe chitosan polymer may be labeled with a fluorophore. In anotherembodiment, either or both of the hydrophilic dispersing agent and thechitosan polymer may be labeled with a fluorophore and also aparamagnetic chelate (chelator) having an MRI (magnetic resonanceimaging) contrast agent bound therein linked to the chitosan polymer sothat the recovered stabilized chitosan-based nanoparticles are effectiveas a bimodal agent that is fluorescent as well as paramagnetic. The MRIcontrast agent may comprise a paramagnetic ion selected from one or moreof gadolinium, dysprosium, europium, and compounds, or combinationsthereof, for example. In one embodiment, the paramagnetic ion comprisesa gadolinium ion and the chelator is a DOTA-NHS ester(2,2′,2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-14,7,10-tetraazacyclododecane-1,4,7-tryl)triaceticacid).Gd³⁺ ions are paramagnetic and DOTA is a chelator of Gd ion. The Gd-DOTAis paramagnetic agent and it provides MRI contrast. Gd-DOTA iscommercially available under the brand name ProHance® (also calledGadoteridol). In another embodiment, either or both of the chitosanpolymer or the hydrophilic dispersing agent may be solely oradditionally linked with iohexyl such that the recovered nanoparticlesare radio-opaque.

A particular embodiment of a DNCP having a Qdot core and achitosan-based shell is shown in FIG. 14. It is appreciated that theillustrated embodiment is merely exemplary and that the nanoparticlesmay include more or less chemical entities than shown, or may compriseentirely different chemical entities. In the illustrated embodiment, theDNCP comprises a Qdot core, which may be any quantum dot material asdescribed above, such as CdS:Mn/ZnS or ZnS:Mn/ZnS. To the Qdot surface,there is attached a N-acetylcysteine (NAC)-modified STAT-3 inhibitor.N-acetylcysteine acts a Qdot surface passivator, as well as a linkerbetween the STAT-3 inhibitor and the Qdot core through its carboxyland/or thiol groups.

The chitosan-based shell comprises chitosan polymer (commerciallyavailable; had a measured molecular weight=approximately 5.3×105 Da; adegree of acetylation=77) and PGA (commercially available, MWapproximately 4130 Da, biocompatible/biodegradable). The chitosanpolymer includes a plurality of amine groups, which will interact byelectrostatic or charge attraction (or otherwise) with the carboxylgroups of the PGA to form an entangled network of the two polymers. Inthe embodiment shown, the PGA and chitosan polymer are cross-linkedusing a water-soluble carbodiimide cross-linker(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC). Alternatively,any other cross-linking compound may be utilized. EDC couplingconjugates the amine groups of the chitosan polymer and the carboxylgroups of the PGA together forming an amide bond.

In the illustrated embodiment of FIG. 14, two imaging agents (AlexaFluor® 660 and Gd-DOTA) are bonded to the chitosan polymer. Alexa Fluor®660 is a commercially available, amine-reactive, near infrared(NIR)-emitting fluorescent dye (MW approximately 1300; wavelength ofabsorption and emission band maxima are 663 nm and 690 nm, respectively;molar extinction coefficient 132,000 cm-1 M-1). Gd-DOTA is acommercially available amine-reactive paramagnetic Gd-chelate complex,which is typically used as a MRI T1 contrast agent. In addition, to thechitosan polymer, there is bonded a folate targeting agent having bothamine and carboxyl functional groups.

In one embodiment, the DNCPs of FIG. 14 may be synthesized by formingthree different W/O microemulsions (ME-I, ME-II and ME-III). Eachmicroemulsion comprises an oil, a surfactant, and an aqueous phasehaving a chitosan polymer. Each microemulsion, however, includes adifferent aqueous phase. The ME-1 aqueous phase comprises Qdots,NAC-conjugated STAT-3 inhibitor (NAC-Drug) and chitosan polymer. Theaqueous phase of ME-II will contain two components, Alexa Fluor® 660 NIRdye conjugated to chitosan polymer (chitosan-dye) and DOTA-Gd (III)conjugated-chitosan polymer (chitosan-DOTA-Gd). The ME-III aqueous phasewill contain EDC cross-linker and folate-conjugated PGA (PGA-folate) andPGA polymer. In an exemplary embodiment, an ME composition contains 6.0mL Triton X-100 (neat), cyclohexane (11.0 mL), n-hexanol (4 mL), and 4.0mL water (i.e. aqueous phase). It is noted that ME-I, ME-II and ME-IIIvary with respect to their aqueous phase contents.

The surfactant may be any suitable surfactant known in the art. Inparticular embodiments, the surfactant comprises a non-ionic surfactant,such as Triton X-100 (e.g., an octylphenol ethylene oxide condensate(P-octyl polyethylene glycol phenyl ether)), available from UnionCarbide, USA. Alternatively, the surfactant may be any other suitablesurfactant material, such as a fatty acid ester, a polyglycerolcompound, a polyoxyethylene surfactant, e.g., asBrij-30, Brij-35,Brij-92, Tween-20, and/or Tween-80. In one embodiment, themicroemulsions may also comprise a co-surfactant. In a particularembodiment, the co-surfactant comprises n-hexanol. N-hexanol is believedto stabilize the interface between oil and water along with the primarysurfactant. In another embodiment, the co-surfactant comprises sodiumbis(2-ethylhexyl)sulfosuccinate (docusate sodium), also soldcommercially as Aerosol® OT (AOT). The oil may be any hydrophobiccompound, such as one that is immiscible with water, e.g., aliphatic andaromatic hydrocarbons. Non-limiting examples of suitable oils for use inthe present invention, e.g. in the first and second microemulsions,include aliphatic and aromatic hydrocarbons, e.g., hexane, heptane,cyclohexane, toluene and benzene. In a particular embodiment, the oilcomprises cyclohexane. The water (aqueous phase) to surfactant molarratio may be any suitable ratio appropriate for the particular materialsand application, such as from about 2:1 to about 70:1, and in aparticular embodiment about 22:1.

To form the DCNPs, Me-II was added to ME-1 followed by addition ofME-III to the mixture. In ME-I, Qdots will be covalently conjugated toNAC-Drug and the resulting conjugate will be coated with chitosanpolymer (DNCP core). The ME-II aqueous components will further coatQdots and protect drugs. EDC will cross-link chitosan and PGA polymers,forming a stable polymeric shell (DNCP shell). Due to the confinedenvironment of the water droplets, the resulting DCNP size is expectedto be about 30 nm.

The produced DNCPs may offer one or more of the following advantages:(i) imageable by both MRI and NIR modalities; (ii) targetable to tumorcells over-expressing folate receptors; (iii) therapeutic as it carriesStat3 SMI (small molecule inhibitor drugs); (iv) particles are highlyhydrophilic and stable in phosphate buffer due to their hybrid nature(chitosan-PGA); (v) biocompatible and stable (cross-linked) DNCP shellstructure (biodegradation rate is expected to be quite slow due tocross-linking); (vi) non-heavy metal based DNCP core when non-cadmiumbased ZnS:Mn/ZnS core is selected; (vii) Improved FRET performance isexpected for CdS:Mn/ZnS Qdots over ZnS:Mn/ZnS Qdots (as CdS:Mn/ZnS Qdotsis excited

efficiently at longer wavelength); (viii) DNCP with CdS:Mn/ZnS core maynot exhibit cytotoxicity as the core is well protected by thecross-linked DNCP shell; and (ix) hundreds of SMIs (small moleculeinhibitors) will be captured in a single particle and they will remainprotected from the adverse extra-cellular environment.

The DNCPs are also porous and hydrophilic particles. When the particlesare administered to a subject, and thereafter internalized and exposedto the cytosolic glutathione (GSH) environment, the disulfide bondbetween the active agent and the Qdot may be cleaved by GSH, resultingin the release of the active agent. The rate of release of the activeagent will depend on several factors, including the diffusion rate ofglutathione, concentration of intra-cellular glutathione, interaction ofdrug to polymeric shell, and the like. It is highly feasible toestablish a sustained drug release mechanism by controlling thethickness of the DNCP shell and degree of cross-linking.

The Qdot fluorescence in the DNCP will be quenched due to an electrontransfer process between the nanoparticles' Qdot core and the Qdot'sligands, e.g., active agents. The restoration of Qdot emission willoccur once drugs are released from Qdot surface upon interaction withintra-cellular glutathione. In one embodiment, the Qdot and the AlexaFluor® 660 are present together in the same nanoparticle and arepurposely selected to form a FRET pair where the Qdot will serve as adonor (600 nm emission) and the Alexa Fluor® 660 (663 nm excitation and690 nm emission maxima) will serve as an acceptor. In absence ofglutathione, Qdot emission is in the “OFF” state, but Alexa Fluor®emission is in the “ON”

state when excited at 663 nm (therefore, trackable by NIR imaging invivo). The quantification of drug release, for example, may be based ona FRET scheme with Qdot core as the donor and the Alexa Fluor® NIR dyeas the acceptor under Qdot excitation. The ratio of emission intensityof Alexa Fluor® 660 (measured at 690 nm) to Qdot (measured at 600 nm)may be directly correlated to the total amount of drugs present in DNCPs(actual concentration of drugs can be quantified by treating DNCPs withexcess amount glutathione and measuring drug fluorescence).

It is appreciated that measure of emission intensities at Alexa Fluor®660 and Qdot peak emission wavelengths is expected to be appropriate tominimize spectral cross-talk while maximizing signal to backgroundratio. The ratio of emission intensity of Alexa Fluor® to Odot willlikely increase drastically once drug is released from the DNCPs andFRET occurs from Qdots to Alexa Fluor®. It is noted that only drugmolecules are expected to escape from the DNCP and both Qdot and AlexaFluor® 660 will remain integrated within the particle in thechitosan-based shell. The amount of drug release can be quantified insolution experiments, for example, by constructing a calibration curveof the above intensity ratio versus drug concentration. This calibrationcurve can be used to estimate drug release in vivo(semi-quantitatively).

As set forth in Table 1 below, several characterization techniques maybe utilized to evaluate DNCPs. Without limitation, these techniquesinclude transmission electron microscopy (TEM), scanning electronmicroscopy (SEM), dynamic light scattering (DLS), UV-visible (UV-VIS)spectroscopy, Fourier transform infrared spectroscopy (FTIR), zetapotential, high pressure liquid chromatography-mass spectrometry(HPLC-MS), NMR/IR, mass spectrometry, fluorescence excitation andemission spectroscopy and fluorescence microscopy, near infrared (NIR)imaging (NIRS), and magnetic resonance (MR) imaging and spectroscopy.Exemplary uses for each technique with respect to the nanoprobesdescribed herein are set forth in Table 1 below.

TABLE 1 Tests Purposes 1. Fluorescence 1. To perform systematicphotophysical characterization of DNCP at (excitation and emission)different stages of development including characterization of drugspectroscopy loading and release 2. Fluorescence Confocal 2. To performmicroscopic imaging of DNCP internalized cancer microscopy cells tolocalization of particles and their fate in intracellular environment 3.Electron microscopy 3. To evaluate DNCP size and morphology(transmission/scanning) 4. Dynamic light 4. To evaluate DNCP sizedistribution and to study their state of scattering dispersion inphosphate buffer. 5. Zeta Potential 5. To evaluate DNCP surface chargeMeasurement 6. UV-VIS measurement 6. To characterize absorption of DNCP7.NMR/IR spectroscopy 7. To characterize Stat3 inhibitor, Qdot-NAC-Stat3 conjugate, EDC based covalent coupling (cross-linking) 8. FTIR 8. Tocharacterize chemical structure using infrared (IR) spectroscopy 9.HPLC-MS 9. To characterize drug loading 10. Mass Spec 10. Tocharacterize Stat3 inhibitor construct 11. NIR Imaging 11. To performoptical based in vivo tumor imaging at NIR region 12. MR imaging and 12.To determine T1, T2 and T2* for agar phantom embedded with spectroscopyDNCP loaded cancer cells as well as tumor tissue

The present inventors have undertaken a successful synthesis of DNCPthat comprise Qdot-STAT3 small molecule inhibitor (SMI) conjugates andhave observed Qdot fluorescence quenching, SMI fluorescence quenching,Qdot and STAT3 restoration upon treatment with glutathione, and folatereceptor-mediated specific uptake as with the MMCNPs set forth above.Further, the present inventors have observed Qdot fluorescencerestoration of the DCNPs in MDA-MB-231 cells upon interaction with theintra-cellular glutathione. A few exemplary benefits and uses of theDNCPs are further summarized in Table 2 below.

TABLE 2 Objectives Approach i) Specific delivery i) Via folate-receptormediated targeting of anticancer drugs to tumor cells ii) Promoting drugii) By encapsulating drugs within DNCP core stability iii) Trackingdrugs iii) By MRI (Gd-DOTA) and NIR (Alexa Fluor 660) imaging of DNCP invivo iv) Confirmation of iv) Drug conjugated Q dots are in fluorescentlyquenched (“OFF”) state due drug release in to electron/energy transferprocess. Drug release process will restore Qdot vivo fluorescence (“ON”state). The “ON” state Qdot will turn on NIR emission of Alexa Fluorthrough fluorescence resonance energy transfer (FRET) process. v)Quantification of v) By measuring ratio of fluorescence intensity ofAlexa Fluor (at 690 nm) drug concentration to Qdot (at 600 nm). Theratio will increase with the release of drugs from within tumor tissueDNCP. vi) Monitor tumor vi) by measuring volume of MRI as well as NIR 3Dimage contrast volume) size vii) Track tumor vii) By MRI and NIRimaging. High T1 relaxivity of DNCP is the key and cells tracking of afew tumor cells together is feasible. Bright NIR emission from AlexaFluor will minimize tissue scattering and auto-fluorescence, thusimproving optical imaging sensitivity down to a cluster of tumor cells.viii) Monitor tumor viii) Again by MRI and NIR imaging. Due to high MRIand optical sensitivity metastasis of the DNCP, imaging of metastatictumor cells is highly feasible. For monitoring tumor metastasis over aperiod of time, multiple administration of DCNP buffer formulation willbe required.

According to another embodiment, the invention pertains to a nanoprobecomprising an inorganic core or an inorganic/Qdot hybrid core. Either ofthese core components are associated with an activatable active agent.In one embodiment, the active agent is one that will be released fromthe core upon exposure to an endogenous molecule, such as glutathione.The nanoprobe is also at least partially or fully encased in a lipidvesicle. In a more specific embodiment, the lipid vesicle isfunctionalized to promote cellular uptake. Non-limiting examples offunctionalization, e.g., surface functionalization, is the linkage offolic acid and/or TAT-peptide to the lipid vesicle.

A non-limiting list of other phospholipids that may be used to formlipid vesicles includes but is not limited to, one or more ofhydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine(EPC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG),phosphatidylinsitol (PI), monosialogangolioside, spingomyelin (SPM),distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine(DMPC), or dimyristoylphosphatidylglycerol (DM PG). In certainembodiments of the invention, the ratio of pharmaceutical agent tolipid-protein ranges from about 0.0005 to about 1 (w/w), more preferablyabout 0.0005 to about 0.5 (w/w), more preferably about 0.001 to about0.1 (w/w).

Phospholipids preferably form an important part of liposomes.Phospholipids are, in their simplest form, composed of glycerol bondedto two fatty acids and a phosphate group. The resulting compound calledphosphatidic acid contains a region (the fatty acid component) that isfat-soluble along with a region (the charged phosphate group) that iswater-soluble. Most phospholipids also have an additional chemical groupbound to the phosphate. For example, if the phosphate is connected withcholine; the resulting phospholipid is called phosphatidylcholine, orlecithin. Other phospholipids include phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine.The fat-soluble portions associate with the fat-soluble portions ofother phospholipids while the water-soluble regions remain exposed tothe surrounding solvent. The phospholipids of the cell membrane forminto a sheet two molecules thick with the fat-soluble portions insideshielded on both sides by the water-soluble portions. This stablestructure provides the cell membrane with its integrity.

The components of liposomes determine the physical characteristics ofthe liposome. Liposomes preferably consist of amphipathic lipidmolecules, with phospholipids being the major component. Most commonly,phosphatidylcholine is used as the primary constituent. Other lipids,including phosphatidylethanolamine, phosphatidylserine, sphingomyelin,glycolipids and sterols are often added. The physical characteristics ofliposomes depend on pH, ionic strength and phase transitiontemperatures. The phase transition consists of a closely packed, orderedstructure, called as the gel-state, to a loosely packed, less-orderedstructure, known as the fluid state. The phase transition temperature(T_(a)) depends on the acyl chain length, degree of saturation, andpolar head group. For example, the T_(c) of egg phosphatidylcholine witha high degree of unsaturation of the acyl chains and varying chainlength is −15 degrees C. However, in a fully saturateddistearoylphosphatidylcholine (DSPC), T_(c) is over 50 degrees C. Mostliposomal formulations contain cholesterol in order to form a moreclosely packed bilayer system during preparation. Cholesterol additionto phosphatidylcholine changes the melting behavior of the bilayer, ascholesterol tends to eliminate the phase transition. Cholesteroladdition has a condensing effect on the fluid-state bilayer and stronglyreduces bilayer permeability.

The fusion of lipid vesicles has been demonstrated to be a powerfulapproach to create a continuous and fluid lipid membrane on planar solidsubstrates. (Sackmann, E., Supported Membranes: Scientific and PracticalApplications. Science 1996, 271, 43.) Recently, there are severalreports on the formation of lipid bilayers on nanoparticles by thefusion of small unilamellar vesicles. (e.g., Cauda, V.; Engelke, H.;Sauer, A.; Arcizet, D.; Brauchle, C.; Radler, J.; Bein, T.,Colchicine-Loaded Lipid Bilayer-Coated 50 Nm Mesoporous NanoparticlesEfficiently Induce Microtubule Depolymerization Upon Cell Uptake. NanoLetters 10, 2484; Li, P. C.; Li, D.; Zhang, L. X.; Li, G. P.; Wang, E.K., Cationic Lipid Bilayer Coated Gold Nanoparticles-MediatedTransfection of Mammalian Cells. Biomaterials 2008, 29, 3617; or Mornet,S.; Lambert, O.; Duguet, E.; Brisson, A., The Formation of SupportedLipid Bilayers on Silica Nanoparticles Revealed by CryoelectronMicroscopy. Nano Letters 2005, 5, 281) It has been shown that thestructure of nanoparticle-supported lipid bilayers is similar to thatformed on planar substrates. The coating of lipid bilayers include theenhancement of the circulation time, accumulation of nanoparticles incells and the minimization of the toxicity of nanoparticles.

In a specific embodiment, the invention pertains to a method ofproducing a nanoprobe encased in a lipid vesicle. In a more specificembodiment, CdS:Mn/ZnS Qdots are synthesized in a modular fashionfollowing three steps.

In a first step, unmodified (bare) Qdots are synthesized using a dioctylsulfosuccinate sodium salt (AOT)/heptane/water microemulsion system asdescribed in the literature. (Santra, S.; Yang, H. S.; Holloway, P. H.;Stanley, J. T.; Mericle, R. A., Synthesis of Water-DispersibleFluorescent, Radio-Opaque, and Paramagnetic Cds: Mn/Zns Quantum Dots: AMultifunctional Probe for Bioimaging. Journal of the American ChemicalSociety 2005, 127, 1656; Santra, S.; Yang, H.; Dutta, D.; Stanley, J.T.; Holloway, P. H.; Tan, W. H.; Moudgil, B. M.; Mericle, R. A., TatConjugated, Fitc Doped Silica Nanoparticles for Bioimaging Applications.Chemical Communications 2004, 2810)

In a second step, S3I drug (containing a secondary nonfunctional aminegroup) will be chemically linked to NAC using standard EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, water soluble) couplingchemistry (as described in reference (Santra, S.; Liesenfeld, B.; Dutta,D.; Chatel, D.; Batich, C. D.; Tan, W. H.; Moudgil, B. M.; Mericle, R.A., Folate Conjugated Fluorescent Silica Nanoparticles for LabelingNeoplastic Cells. Journal of Nanoscience and Nanotechnology 2005, 5,899)) to obtain the NAC-S3I conjugate. A 1:1 molar ratio of NAC:S3I maybe used to avoid the presence of excess NAC in the reaction mixture. Ina previous paper, small lipid vesicles of Zwitterionic1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC)were synthesized by dissolving lipid molecules in water at aconcentration of 2 mg/mL. The lipid solution was incubated at 50 degreesC. for 3 hours and then extruded 10 times through a Nucleopore membraneusing a Lipex extruder. FIG. 19a shows an AFM image of resultingvesicles.

Lastly, the NAC-S3I conjugate is reacted with the unmodified Qdots toobtain S3I surface conjugated Qdots. The product, Qdot-S3I conjugates,may be separated from the microemulsion system by thorough washing with95% ethanol. These particles are then be further surface modified with alipid bilayer (LB) to obtain LB coated QDot-S3I. Upon reaction withintracellular GSH, the disulfide bond between the QDot core and NAC-S3Iconstruct is cleaved, thus releasing the drug from the QDot surface andresulting in restoration of quenched Qdot fluorescence in proportion toamount of drug released, as schematically illustrated in FIG. 17.

As described above, the nanorpobes may be coated with lipid bilayersthrough the fusion of small unilamellar vesicles. In a specificembodiment, Zwitterionic lipid (DC8,9PC), positively charged lipid1,2-dioleoyl-3-rimethylammonium-propane (DOTAP) (FIG. 19b ), and1-hexadecanoly,2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphatidylethanolamine(PtdEtn)(FIG. 19c ) is used. First, small unilamellar vesicles ofDC8,9PC, DOTAP and PtdEtn with diameters in the range of 15-50 nm areprepared by extrusion of hydrated lipid films through filters with apore size at the nanometer scale with standard protocols. (Mayer, L. D.;Hope, M. J.; Cullis, P. R., Vesicles of Variable Sizes Produced by aRapid Extrusion Procedure. Biochimica Et Biophysica Acta 1986, 858,161). The lipid bilayers obtained from PtdEtn or DOTAP/PtdEtn mixturescan be further modified with folic acid or TAT-peptide by covalentlylinking to the amine group of PtdEtn. Second, the zeta potential oflipid bilayer-coated Qdots is measured and small unilamellar vesicles toconfirm the fusion on Qdots. Third, the formation of lipid bilayers onQDots is observed with cryotransmission electron microscope, which hasbeen used in imaging the lipid bilayers on silica nanoparticles. (Liu,J. W.; Jiang, X. M.; Ashley, C.; Brinker, C. J., ElectrostaticallyMediated Liposome Fusion and Lipid Exchange with aNanoparticle-Supported Bilayer for Control of Surface Charge, DrugContainment, and Delivery. Journal of the American Chemical Society2009, 131, 7567). The resulting biosensor particles are shown in FIGS.18a-b . In particular, FIGS. 18a-b shows a nanoparticle 10 having aCdS:Mn/ZnS quantum dot core 12, to which an S3I inhibitor drug 14 willbe covalently linked via a cleavable disulfide bond linkage. (a) a lipidbilayer 16 may be overcoated on the surface of the S3I conjugatedCdS:Mn/ZnS quantum dots. (b) the lipid bilayer may be furtherfunctionalized with folic acid 18 as shown in FIG. 18b to target cancercells that overexpress folate receptors, or with TAT peptide (acellpenetrating peptide).

In an alternative embodiment, the invention pertains to an activatablenanoprobe. The nanoprobe embodiment comprises a core component that isassociated with an active agent. The core component may comprise aninorganic core component. Alternatively, the core component may comprisean inorganic/Qdot hybrid core. The active agent may be associated with alinker comprising a double sulfide bond. The construction of thenanoprobe allows for activation of the active agent when exposed to anendogenous compound such as GSH. Thus, certain nanoprobe embodiments donot necessarily incorporate the use of a Qdot or similar opticallydetectable means or structure.

The nanoprobes, ligands and compounds described herein may be providedin an effective amount, namely an amount effective to achieve thedesired result.

The following examples are intended for the purpose of illustration ofthe present invention. However, the scope of the present inventionshould be defined as the claims appended hereto, and the followingexamples should not be construed as in any way limiting the scope of thepresent invention.

Example 1

The following example more particularly describes the production of anexemplary quantum dot (Qdot)-iron oxide (10) basedmultimodal/multifunctional nanocomposite probe (MMCNP) that is opticallyand magnetically imageable, targetable, and capable of reporting onintracellular drug release events.

1.1 Materials

All the chemicals were used as received. Ferric (III) chloridehexahydrate, Ferrous (II) chloride tetrahydrate, ethylenediamine werepurchased from Fluka. Lipoic acid and sodium borohydride were purchasedfrom Sigma-Aldrich. Methyl-PEG-12-NHS, and folic acid were purchasedfrom Fisher Scientific, USA. All the other chemicals and solvents werealso purchased from Fisher Scientific, USA. Nanopure water (deionizedand filtered water) was used for the following example. Fluorescencespectra were recorded in NanoLog Spec Fluorimeter, Perkin Elmer. TheFTIR spectra were recorded in a Perkin Elmer Spectrum 100. UV-Visspectra were recorded in a Cary Win UV spectrometer. The low and highresolution electron microscopic images were taken in TEM JEOL 1011 andFEI Tecnai F30 TEM instruments respectively.

The STAT-3 inhibitor used was an SF-1-046 drug. The non-phosphorylatedsalicylic acid-based small-molecule, SF-1-046, belongs to theS31-201.1066 class of STAT3 inhibitors^(31,32). Compounds in this class,including SF-1-046, are structural analogs of the previously reportedlead STAT-3 dimerization disruptor, S3I-201³³. Consistent with thepublished reports regarding the activities of the lead and the othermembers of the second generation class of compounds³¹⁻³³, GOLDcomputational modeling³⁴ indicated SF-1-046 interacts with the STAT3 SH2domain (data not shown), disrupting STAT3 SH2 domain:pTyr interactions,and thereby inhibits STAT-3 activation. SF-1-046 was prepared viapreviously published synthetic protocols^(31,32).

1.2 Methods (Synthesis and Surface Modification of Iron OxideNanoparticle Cores (IONPs) by Dihydrolipoic Acid)

Super paramagnetic iron oxide nanoparticles (IONPs) were preparedfollowing a previously established protocol³⁵. In brief, 1.0 M (1.13 g)of ferric chloride hexahydrate (FeCl₃.6H₂O); 0.5 M (0.415 g) ferrous(II) chloride tetrahydrate (FeCl₂.4H₂O); 0.177 mL of 37% HCl in 4 mL ofDI water were taken and stirred vigorously in a vortex in 15 mLcentrifuge tube. 1.66 mL of 28-30% NH₄OH solution was dissolved in 31 mLof DI water in a conical flask and stirred vigorously for 5 min.Thereafter, the ferric chloride/ferrous chloride/HCl solution wassuddenly added to the stirring ammonia solution, and the mixture wasstirred again for 30 min with 800 rpm at room temperature in a nitrogenatmosphere to prevent critical oxidation. The black precipitate formedinstantly. After complete stirring the iron oxide nanoparticles wereseparated by sedimentation at the bottom of the flask using an externalneodymium magnetic field. 15 mL of the supernatant was decanted and thenagain the stirring was continued at 800 rpm. 80 mg of lipoic acid (LA)was added in 20 mL chloroform and was vortexed for 20 min. This lipoicacid solution was then added to the iron oxide nanoparticle dispersionshortly. The lipoic acid was used to coat the iron oxide nanoparticles(FIG. 12). After 4h of complete stirring, 20 mL of methanol was added toreduce the viscosity of the aqueous phase and allowed the chloroformbased ferrofluid to settle at the bottom of the container. The clearaqueous phase was first decanted and then the chloroform part wasdiluted again by 10 mL of chloroform. The chloroform residue was thentaken in a separating funnel and washed with water to neutrality. Thechloroform part then taken in a 50 mL conical vial and dried undervacuum to get the black solid powder. Thus, lipoic acid-coated ironoxide nanoparticles (LA-IO) were obtained.

To chemically reduce the disulfide bond of LA-IO to dithiol groups, 0.2g of solid black lipoic acid was coated iron oxide nanoparticles in 50mL of water/ethanol mixture (1:1), then the dispersion wasultra-sonicated for 10 min, followed by vigorous stirring for another 20min in ice bath. 0.2 g of ice cold solution of freshly prepared sodiumborohydride was slowly added to it under vigorous stirring conditions.After complete addition of the solution, the stirring was continued foranother 2 h. The black materials were then separated by sedimentationusing a strong neodymium magnet and washed with DI water to neutrality.The material was then taken in choloroform and dried under vacuum to getthe solid black dihydrolipoic acid coated iron oxide nanoparticles(DHLA-IO). They were used for the next step of reaction as synthesized.

1.3 Synthesis of CdS:Mn/ZnS Quantum Dots (Qdots).

Dopant based core-shell CdS:Mn/ZnS nanocrystals were used in thisexample. The CdS:Mn/ZnS Qdots were synthesized by a water-in-oil (W/O)microemulsion method following a published protocol³⁶. In brief, cadmiumacetate dihydrate (Cd(CH₃COO)₂.2H₂O), manganese acetate tetrahydrate(Mn(CH₃COO)₂.4H₂O), sodium sulfide (Na₂S), and zinc acetate dihydrate,metal basis (Zn(CH₃COO)₂.2H₂O) were used for the preparation of Cd²⁺ andMn²⁺; S²⁻; and Zn²⁺ ion-containing standard aqueous solutions. Theaqueous solution was stirred for 15 min and then added to theAOT/heptane solutions to form the water-in-oil (W/O) microemulsions. TheMn-doped CdS core nanocrystals were formed by mixing (Cd²⁺ and Mn²⁺) andS²⁻ containing (W/O) microemulsions rapidly for 10-15 min. The W₀(water-to-surfactant ratio) value of W/O microemulsions were maintainedat 10. For the growth of outer shell layer on the Mn doped CdS coreQdots, the Zn²⁺ ion containing (W/O) microemulsion was added at veryslow rate (1.5 mL/min) to the (W/O) microemulsions containing CdS:Mn.The nucleation and growth of a separate ZnS phase were suppressed by thevery slow addition of the Zn²⁺ containing W/0 microemulsion. The [Zn²⁺]to [Cd²⁺] molar ratio (X₀) was 8 for our study.

1.4 Synthesis of NAC Derivatives of Folic Acid (FA), Drug (STAT3Inhibitor) and Ethylenediamine (EDA).

Folic acid, drug (STAT3 inhibitor) and ethylenediamine (EDA) wereseparately conjugated to NAC following standard bioconjugationtechniques as described below.

a) STAT3-NAC Conjugation:

A 2 mL anhydrous DMSO solution containing 3×10⁻⁵ mol of N-acetylcysteine, 5.7×10⁻⁴ mol of EDC, and 1.5×10⁻⁴ mol of NHS was stirred for30 min at room temperature. After this incubation, 1.5×10⁻⁵ mol of solidSTAT3 inhibitor compound (contains secondary non-functional amine group)was added and whole solution mixture was stirred for 2 h at roomtemperature. The reaction mixture was passed through 0.2 μm Whatmanfilter membrane and the solution was then dried under vacuum. Theproduct was dispersed in 1 ml nanopure water (water that has beendeionized and then filtered so that no particles greater than 1.0nanometer remain).

b) EDTA-NAC Conjugatation:

Following the same procedure as above for STAT3-NAC, a 2 mL anhydrousDMSO reaction mixture containing 1.2×10⁻⁴ mol of N-acetyl cysteine,1.2×10⁻³ mol of EDC, and 0.3×10⁻³ mol of NHS was stirred for 30 min atroom temperature. After this incubation, 1.2×10⁻³ mol of ethylenediamine(EDTA) was added and the whole reaction mixture was then stirred foranother 2 h at room temperature. Then, N₂ gas was passed through itfollowed by filtration through a 0.2 μm Whatman membrane. This solutionwas then dried under vacuum and the dried product was then dispersed in1 ml nanopure water.

c) Folic Acid-NAC Conjugation:

Following the same procedure as above for STAT3-NAC and EDA-NAC, 2 mLPBS solution containing 2.8×10⁻⁵ mol of folic acid, and 5×10⁻⁵ mol ofEDC was stirred for 30 min at room temperature. To this solution, 0.5 mlof the NAC-EDA complex was added. The resulting reaction mixture wasthen stirred for overnight in dark at room temperature. The reactionmixture was then passed through a 0.2 μm cut-off Millipore® membranefilter. This solution was then dried under vacuum and finally dispersedin nanopure water.

1.5 Synthesis of Qdots Attached to Iron Oxide Nanoparticles(IONPs-Qdots)

In this synthesis process, the CdS:Mn/ZnS core-shell quantum dots(Qdots) were extracted from the microemulsion solution by repeatedcentrifugation followed by washing several times with methanol andethanol. The extracted Qdots were dispersible in DI water and used asextracted from microemulsion solution. To the 1 mL Qdots dispersion (20mg/mL) in water, the DHLA-coated IONP dispersion (2 mg/mL) in 4 mLethanol was added dropwise. After complete addition, the whole reactionmixture was stirred for overnight. Thereafter, the nanoparticles wereseparated by a strong external neodymium magnet and washed several timeswith ethanol and DI water to remove the unused Qdots. In this way, theQdots were attached on the surface of IONPs through a DHLA linker. Theattachment of Qdots on the IONP surface, however, partially reduced thebrightness of the Qdot luminescence. The IONP-Qdot composites werebright enough to be easily visualized under illumination by a hand-held366 nm multiband UV light source. Furthermore, the IONP-Qdot compositesresponded well to external magnetic fields.

1.6 Surface Functionalization of IONPs-Qdots

The 2 mg/mL IONPs-Qdots nanoparticles were taken in DMSO/ethanol (4:1)mixture and stirred, as well as sonicated to be well dispersed. Thisdispersion was showing fluorescence under hand-held 366 nm multiband UVlight source. This dispersion was then added to a mixture of 0.7 ml ofdrug (STAT-3)-NAC, 0.2 ml of EDA-NAC and 0.4 ml of FA-NAC conjugatesslowly under constant vortexing and UV exposure. It was observed thatthe previous fluorescence intensity of the IONP-Qdot conjugates wasquenched. The reaction was stirred for overnight and then the wholeconjugated nanocomposites were separated by a strong external neodymiummagnet and washed several times with DMSO and nanopure water. Finally,the reaction mixture was dispersed in 1 ml of 0.1(M) NaHCO₃ solution andstirred for few minutes in dark. After this stirring, 5.5 mg ofmethyl-PEG-NHS ester was added to it and the whole solution was stirredfor overnight. These activatable multifunctional/multimodal compositenanoprobes (MMCNPs) were then separated by an external neodymium magnetand washed several times with DPBS (Dulbecco's phosphate bufferedsaline), and finally was taken in DPBS for further use.

1.7 Preparation of MRI Samples

Human breast cancer (MDA-MB-231), pancreatic cancer (Panc-1), and mousethymus stromal epithelial (TE-71) cells have all been previouslyreported^(31,32). Cells were grown in Dulbecco's modified Eagles'smedium (DMEM) containing 10% heat-inactivated fetal bovine serum. Cellswere treated with the nanoparticles set forth in section 1.6 above at aconcentration of 0.1 mg/ml for 24 hours The medium was extracted byvacuum, and cells were washed by 1×PBS buffer for 6 times to remove theunbound nanoparticles. Cells were detached by Trypsin with 0.25% EDTA,centrifuged down at 1500 rpm for 3 minutes and then discardedsupernatant. Cells were then resuspended in sterile water and mixed withequal volume 3×PBS and 3% agarose, and carefully poured into 10 mm NMRtubes. Positive control was prepared by mixing 0.3 mg/ml nanoparticlessolution with equal volume of 3×PBS and 3% agarose.

1.8 Magnetic Resonance Imaging

Magnetic resonance imaging of a layered cell phantom was performed at14T magnetic field strength using Paravision 3.0.2 software and a 10 mmmicroimaging coil (Bruker). A three-dimensional gradient echo scansequence (FLASH) was acquired with following settings; repetition time(TR)=200 ms, echo time (TE)=2.7 ms, 128×128×128 matrix size and field ofview (FOV)=10×10×20 mm³. Hypointense signal from clusters of iron oxidecontaining cells, was inverted and assigned a red pseudocolor for imagepresentation on subsequent 3D renderings performed using OsiriX viewingsoftware (http://www.osirix-viewercom).

1.9 CyQUANT™ Cell Proliferation Assay

Human breast cancer (MDA-MB-231) cells, mouse thymus epithelial stromal(TE-71) cells, and pancreatic cancer (Panc-1) cells were grown inDulbecco's modified Eagles's medium (DMEM) containing 10%heat-inactivated fetal bovine serum. 5000 cells per well were culturedin 96-well plates. The cells were treated with nanoparticles, compounds(drug), and nanoparticles conjugated to compound for 24 hours. Cyquantcell proliferation assays (Invitrogen Corp/Life Technologies Corp,Carlsbad, Calif.) were then performed on each microplate well. Themedium was removed by vacuum, and 50 μL 1× dye binding solution wereadded to each microplate well, which were then incubated at 37° C. for30 minutes. The fluorescence intensity of each sample was measured usinga fluorescence microplate reader (POLARstar Omega, BMG Labtech, Durham,N.C., USA) with excitation at about 485 nm and emission detection atabout 530 nm.

1.10 Confocal Fluorescence Microscopy

The cells were fixed on the glass slide after 24 hrs of incubation withthe nanoparticles, compounds (drug), and nanoparticles conjugated tocompound. Confocal fluorescence microscopy on the cells was done with ahome-built sample-scanning confocal microscope. The excitation sourcewas 375 nm pulse diode laser (PicoQuant GmbH, LDH-P-C-375). The powerused was 3nW. The laser was focused on a spot size of ˜300 nm with aZeiss 100× Fluar objective lens (NA 1.3, WD 0.17 mm). The sample wasraster scanned using a piezoelectric stage (Mad City Labs, Nano-LP100)to get the fluorescence images of the cells with the quantum dots. Thefluorescence was detected using the avalanche photodiode (PerkinElmerSPCM-AQR-14). The spectra were collected using a spectrograph with agrating (150 g/mm, blaze: 500 nm) centered at 600 nm (PI Acton SP-2156),which was coupled to a thermoelectrically cooled Electron MultiplyingCharge Coupled Device (EM-CCD Andor iXon EM+ DU-897 BI). Spectra werecollected from different spots on the cells. Each spectrum was collectedwith 10 sec exposure time and with three consecutive exposures. Thesespectra were then averaged in a home-written Matlab program (MathworkInc. Natick, Mass.). After taking 100 averaged spectra, they spectrawere compiled and an ensemble spectrum was built in Matlab program. Thecorresponding bright field images were taken by using the samespectrograph with grating (1200 g/mm, blaze: mirror) centered at 4 nmand coupled with EM-CCD. The exposure time for bright field image was0.05 sec.

1.11 UV-Vis and Fluorescence Measurements

The UV-Vis absorption spectra were collected by using 1 cm path lengthquartz cuvette with an Agilent 8453 spectrometer. The fluorescenceemission spectra were taken by using the 1 cm path length quartzcuvettes with a Nanolog™ Horiba Jobin Yvon fluorometer. The excitationand emission slits were 5 nm. The excitation wavelength was 375 nm forquantum dots and 300 nm for the drug (STAT3 inhibitor (SF-1-046, drug).

In 2 mL of PBS, 100 μL quantum dots in PBS were added and the absorptionand emission was taken. Thereafter, 50 μL of 0.3 M solution of GSH wasadded and absorption and emission spectra were collected immediatelyafter addition of GSH and after every 5 min up to 60 min. This gave aplot of λ_(max) emission vs. time in min. After 35 min, the fluorescencewas nearly constant showing that the drug is completely released after35 min.

Example 2

In this example, two different water-soluble biomolecules, the N-acetylcysteine (NAC) and the glutathione (GSH), were used as surface coatingligands for the Qdot nanoparticles. This includes a single-step,one-part synthesis where the Qdot nanocrystals were grown in thepresence of the biomolecules. These Qdots were characterized byfluorescence spectroscopy. Stability of the GSH-coated Qdots and theNAC-coated Qdots were studied by treating the coated Qdots withethylenediaminetetraacetic acid (EDTA, a strong chelating agent for Znand Cd ions). The results show that fluorescence properties of Qdots areaffected by the type of surface coated ligands. In comparison to theGSH-coated Qdots, the NAC-coated Qdots show broad, but strong emissiontowards near infra-red region. When treated with EDTA, the fluorescenceproperty of the GSH-coated Qdot was affected less than the NAC-coatedQdots. This preliminary study shows that NAC-coated Qdots could thuspotentially be used to develop activatable (“OFF/ON”) probes forpotential deep-tissue imaging applications., the GSH-coated Qdots couldthus be applied for probing desired analytes or for bioimaging purposesin environmentally harsh conditions.

2.1 Preparation of GSH-Coated Qdots

A water-in-oil (W/O) microemulsion technique was used to synthesizeGSH-coated CdS:Mn/ZnS Qdots at room temperature. The W/O microemulsionsystem consisted of dioctyl sodium sulfosuccinate (called Aerosol OT orAOT—a surfactant), heptane (oil) and water (as an aqueous phase).Acetate salts of bivalent cadmium, zinc and manganese were using as anionic source. In a typical procedure, three separate aqueous stocksolutions containing acetate salts were prepared using DI water first;Solution A—10.0 mL aqueous solution containing cadmium acetate dihydrate(258.5 mg), 7.4 mg manganese acetate tetrahydrate and 15 mg of GSH;Solution B—5.0 mL aqueous solution containing 257.5 mg sodium sulfideand Solution C—5 mL aqueous solution containing 285.3 mg zinc acetatedihydrate. Next, 35 mL of AOT stock solution was prepared by dissolving4.46 g of AOT in heptane under magnetic stirring for about 30 mins.Then, solution A1 was prepared by mixing 0.18 mL of stock Solution Awith 5 mL of AOT/heptane solution, Solution B1 was prepared by mixing0.54 mL of stock Solution B with 15 mL of AOT/heptane solution andSolution C1 was prepared by mixing 0.54 mL of stock Solution C with 15mL of AOT/heptane solution.

Solutions A1, B1 and C1 were then magnetically stirred for 1 hour.Solution A1 was then added to Solution B1 and the resulting mixedsolution (Solution AB1) was stirred magnetically for 15 mins. SolutionAB1 was added dropwise (using a burette) at a rate 2-3 mL per min to theSolution C1 under magnetic stirring. To obtain maximum brightness fromQdots, this solution mixture should be stirred for 7 days at the roomtemperature. The GSH-coated CdS:Mn/ZnS Qdots were then isolated from theW/O microemulsion system after precipitating them first using 95%ethanol, followed by repeated washings (6-7 times) with ethanol andethanol-water mixture to remove surfactants, any un-reacted ions andexcess GSH. Ultra-centrifugation technique was used between twosuccessive washing steps to isolate GSH-Qdots in the pellet form fromthe solution. The NAC-coated Qdots were similarly prepared by adding 15mg of NAC in Solution A (in place of 15 mg GSH). All the chemicals,reagents and solvents were purchased from Aldrich-Sigma and used withoutany further purification. The Barnstead Nanopure DI water was used toprepare all the aqueous solutions. Both GSH-Qdots and NAC-Qdots weredispersed well in DI water.

2.2. Fluorescence Study

Both GSH-Qdot and NAC-Qdot exhibited bright yellow emission when exposedto a hand-held UV illumination.

2.3. Excitation of GSH-Qdots

The GSH coated Qdots emits at ˜620 nm upon excitation at 375 nm. Incomparison to the NAC-Qdot, the GSH-Qdots are moderately bright. FIG. 20shows the effect of increase of EDTA concentration on the fluorescenceintensity of GSH-Qdot in DI water at the room temperature. Nosignificant change in the fluorescence intensity was observed.Initially, there was a slight quenching (i.e. decrease in thefluorescence intensity) followed by restoration of fluorescenceintensity to its initial value. This data suggest that EDTA has minimaleffect on the stability of GSH-Qdots, even though EDTA is known to formstable water soluble complex with both the zinc and cadmium ions. TheGSH-Qdot fluorescence is however quenched by the copper ions. FIG. 21shows the effect of increase of Cu ion concentration on the fluorescenceintensity of GSH-Qdots. A steady decrease in the fluorescence intensitywas observed with the increase in the Cu ion concentration. The inset ofthe FIG. 21 shows the linear relationship between the fluorescenceintensity and the Cu ion concentration. While not wishing to be bound bytheory, our results suggest that Cu ions are possibly reacting with theQdots, forming copper sulfide on the Qdot surface. Due to this chemicaltransformation the microenvironment of Mn is altered, resulting in thecreation of surface related defects. Such defects could result in theincrease of non-radiative processes and fluorescence quenching.

2.4 Preparation of NAC-Qdots

The NAC-coated Qdots are highly soluble in DI water and they exhibitextremely bright fluorescence properties. A very broad fluorescenceemission in the range 525 nm-800 nm was observed. This broad emissioncould have been originated due to NAC-induced surface-related defects.FIG. 22 shows the effect of increase of EDTA concentration on thefluorescence intensity of NAC-Qdots. A steady decrease in thefluorescence intensity was observed upon increase in the concentrationof EDTA. This observation suggests that NAC-Qdot is not stable againstthe EDTA chelator. The fluorescence quenching could be due to directbinding of EDTA to the ZnS surface and/or replacement of the NAC by theEDTA. It is well known that EDTA forms a stable complex with Zn ions. Asimilar observation (i.e. steady decrease in the fluorescence intensity)was made when the NAC-Qdot was exposed to the increasing concentrationof Cu ions (FIG. 23). This may be attributed to the formation of Cusulfide on to the Qdot surface.

In summary, we have described a simple but robust method of makingwater-soluble CdS:Mn/ZnS at the room temperature. Both the GSH and theNAC are capable of coating Qdot surface via conjugation through theirsulfhydryl (—SH) groups and thus forming hydrophilic Qdots. The GSHQdots are more stable than NAC-Qdots when challenged against a strongchelator, EDTA. The Cu ions are able to quench fluorescence of both theGSH-Qdot and the NAC-Qdot, suggesting the formation of copper sulfide onto the Qdot surface. The NAC-Qdot emission band is broader than the GSHQdots. Therefore, NAC-Qdots may be used for NIR imaging of biologicaltissues using two-photon excitation. Our study suggests that NAC-Qdotscould be an attractive choice for the fabrication of activatable(“OFF/ON”) Qdots for bioimaging and sensing applications.

While various embodiments of the present invention have been shown anddescribed herein, it will be obvious that such embodiments are providedby way of example only. Numerous variations, changes and substitutionsmay be made without departing from the invention herein. Accordingly, itis intended that the invention be limited only by the spirit and scopeof the appended claims.

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The present application further cross-references US Published PatentApplication Nos. 20110021745, 20100254911, 20100254911, 20070269382,20070264719, 20060228554, each of which is incorporated by referenceherein

The invention claimed is:
 1. A method for monitoring intracellular drugdelivery within a subject comprising: administering to the subject aneffective amount of an optically activatable nanoprobe, the opticallyactivatable nanoprobe comprising: a core component comprising a quantumdot; and an active agent linked to the quantum dot; wherein, uponintracellular uptake of the nanoprobe, a linkage between the activeagent and the core component is cleaved to allow for release of theactive agent and to allow the quantum dot to transfer from a quenchedstate, wherein the luminescence of the plurality of quantum dot isquenched, to a luminescent state, wherein the luminescence of theplurality of quantum dot is activated; and detecting a presence of thequantum dot, wherein an increase in luminescence of the quantum dot isindicative of a release of the active agent intracellularly.
 2. Themethod of claim 1, wherein the linkage between the active agent and thequantum dot is cleavable by intracellular glutathione.
 3. The method ofclaim 1, wherein the nanoprobe further comprises a targeting agent. 4.The method of claim 1, wherein the nanoprobe further comprises ahydrophilic dispersing agent linked to the core component, and whereinthe hydrophilic dispersing agent comprises N-acetyl cysteine.
 5. Themethod of claim 1, wherein the nanoprobe further comprises a hydrophilicdispersing agent linked to the core component, and wherein thehydrophilic dispersing agent comprises glutathione.
 6. The method claim1, further comprising forming a hydrophilic coating about the corecomponent.
 7. The method of claim 1, wherein the active agent comprisesa STAT-3 inhibitor.
 8. The method of claim 7, wherein the active agentfurther comprises N-Acetyl-L-Cysteine directly or indirectly linkedthereto, wherein the core component comprises an inorganic corecomprises iron oxide and a plurality of quantum dots linked thereto, andwherein the plurality of quantum dots comprise CdS:Mn/ZnS quantum dots.9. A method for monitoring intracellular drug delivery within a subjectin whom an effective amount of an optically activatable nanoprobe hasbeen administered, the optically activatable nanoprobe comprising: aninorganic core; a plurality of quantum dots linked to the inorganiccore, and at least one ligand linked to respective ones of the pluralityof quantum dots, the at least one ligand comprising at least an activeagent linked to the quantum dot by a linkage; and wherein, uponintracellular uptake of the nanoprobe, a linkage between the activeagent and a respective quantum dot is cleaved to allow for release ofthe biologically active agent and to allow the plurality of quantum dotsto transfer from a quenched state, wherein the luminescence of theplurality of quantum dots is quenched, to a luminescent state, andwherein the luminescence of the plurality of quantum dot is activated;the method comprising: confirming release of the biologically activeagent by detecting a presence of the plurality of the quantum dot in theluminescent state.
 10. The method of claim 9, wherein the confirming isdone by magnetic resonance imaging.
 11. An activatable nanoprobecomprising: a core component; an activatable active agent associatedwith the core component via a bond configured to be cleaved uponexposure to an endogenous compound.
 12. The nanoprobe of claim 11,wherein the association between the active agent and the core componentis cleavable by intracellular glutathione.
 13. The nanoprobe of claim11, wherein the nanoprobe further comprises a ligand.
 14. The nanoprobeof claim 13, wherein the ligand is a targeting agent.
 15. The nanoprobeof claim 11, wherein the nanoprobe further comprises a hydrophilicdispersing agent linked to the core component, and wherein thehydrophilic dispersing agent comprises N-acetyl cysteine.
 16. Thenanoprobe of claim 11, wherein the nanoprobe further comprises ahydrophilic dispersing agent linked to the core component, and whereinthe hydrophilic dispersing agent comprises glutathione.
 17. Thenanoprobe claim 11, further comprising forming a hydrophilic coatingabout the core component.
 18. The nanoprobe of claim 11, wherein theactive agent comprises a STAT-3 inhibitor.
 19. The nanoprobe of claim18, wherein the active agent further comprises N-Acetyl-L-Cysteinedirectly or indirectly linked thereto, wherein the core componentcomprises an inorganic core comprises iron oxide and a plurality ofquantum dots linked thereto, and wherein the plurality of quantum dotscomprise CdS:Mn/ZnS quantum dots.